Lyophilized Compositions of Phenobarbital Sodium Salt

ABSTRACT

A composition of, method for producing, and use of an amorphous lyophilized Phenobarbital Sodium having high purity are presented. The amorphous lyophilized Phenobarbital Sodium is storage-stable being essentially void of impurities (e.g., phenylethylacetylurea (PEAU), 2-ethyl-2-phenylmalonamide (2EPMM), and/or alpha-phenylbutyrylguanidine (PBG)) upon reconstitution in water.

RELATED APPLICATIONS

This application is a continuation application of our co-pending U.S.application with the Ser. No. 17/854,914, filed Jun. 30, 2022, which isa continuation-in-part application of our U.S. application with the Ser.No. 17/025,881, filed Sep. 18, 2020, now U.S. Pat. No. 11,406,598, whichclaims the benefit of U.S. Provisional Patent Application No.62/903,511, filed on Sep. 20, 2019, which is incorporated by referencein its entirety.

FIELD OF THE INVENTION

The present disclosure relates to compositions of storage-stablelyophilized Phenobarbital Sodium having high purity that are essentiallyvoid of impurities upon reconstitution in water.

BACKGROUND OF THE INVENTION

The background description includes information that may be useful inunderstanding the present disclosure. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

All publications and patent applications herein are incorporated byreference to the same extent as if each individual publication or patentapplication were specifically and individually indicated to beincorporated by reference. Where a definition or use of a term in anincorporated reference is inconsistent or contrary to the definition ofthat term provided herein, the definition of that term provided hereinapplies and the definition of that term in the reference does not apply.

Phenobarbital Sodium is a well-known barbiturate that is most commonlyadministered as an anti-convulsant for the treatment of seizures and mayalso be administered as a sedative, a sleep aide, or a pre-anesthetic.Phenobarbital Sodium is typically a liquid formulation administeredintramuscularly (IM) or intravenously (IV) with crystalline granules orcrystals dissolved in an aqueous solvent. For example, WO 2017/085687 toParker et al. discloses a liquid formulation of Phenobarbital Sodiumdissolved in water along with a C1-C4 alcohol and a glycol.

The stability of solubilized Phenobarbital Sodium has been a recurringproblem because it readily hydrolyzes in water forming variousdegradation products. In particular, in water alone or in water with anincrease in pH, Phenobarbital Sodium forms the degradation productsphenylethylacetylurea (PEAU), 2-ethyl-2-phenylmalonamide (2EPMM), andalpha-phenylbutyrylguanidine (PBG). While these degradation impuritiesmay not directly compromise the effectiveness of the PhenobarbitalSodium for its therapeutic activity, these degradation impurities aretoxic and cause severe side effects. Specifically, PEAU was previouslyadministered as Pheneturide for severely affected epileptics. However,the Food and Drug Administration (FDA) withdrew approval of Pheneturidedue to its toxic effects. In particular, Pheneturide is reported to be apotent liver enzyme inducer that disrupts both calcium and folatemetabolism. (Nayyar et al., 2012, J. of Appl. Pharm Sci., 2:230-235.)Prior to its removal as an FDA-approved drug, patients treated withPheneturide showed low levels of serum calcium and folate.

Over the years of medical administration, efforts have been made todecrease the formation of the toxic degradation products in thePhenobarbital Sodium liquid formulation. For example, Gupta, V. D., (J.Pharm Sci., 1984, 73:1661-1662) describes the effects of ethanol,glycerol, and propylene glycol on the stability of Phenobarbital Sodiumin aqueous solutions. Specifically, Gupta discloses that ethanol has thebest effect followed by propylene glycol and glycerol relative to wateralone. Years after Gupta, Parker et al., (WO 2017/085687) focused onminimizing the total amount of water to reduce the hydrolysis andconsequently the formation of the degradation products. Parker addressesthe hydrolysis issue by limiting the amount of water (e.g., to no morethan 50 mg/mL) in the liquid formulation. Parker's minimized waterformulations also include a C1-C4 alcohol such as ethanol and a glycolsuch as propylene glycol. However, while the amount of impurities inParker's liquid formulation may have decreased compared to priorformulations, impurities such a PEAU are still present in undesirableamounts, particularly after extended storage.

In addition to the degradation impurities, the current marketed liquidformulation of Phenobarbital Sodium (e.g., Phenobarbital Sodium forInjection USP, 65 mg/mL or130 mg/mL), includes organic solvents such asalcohol, which are not desirable in general and indeed not suitable foradministration to newborns as described in the art, e.g., Williams etal., 1998, “Evaluating Toxic Alcohol Poisoning in the EmergencySetting,” Laboratory Medicine, Vol. 29, No. 2; Wood et al., 2007, J. ofPerinatology, 27:183-185; Kumar, 1985, “Adverse Drug Reactions in theNewborn,” Annals of Clin. And Lab. Sci, Vol. 15, No. 3; and Cuzzolin,2018, J. Pediatric and Neonatal Individualized Med., doi:10.7363/070112.

Accordingly, there is still a need for a stable Phenobarbital Sodiumformulation in a formulation that is storage stable and void oressentially void of degradation impurities upon solubilization in water.

SUMMARY

Disclosed herein are compositions of, methods of producing, and use of alyophilized Phenobarbital Sodium formulation. The contemplatedcomposition is an amorphous lyophilized Phenobarbital Sodium with highstability and purity. The lyophilized composition may include no lessthan 98% Phenobarbital Sodium. The amorphous composition is astorage-stable form of lyophilized Phenobarbital Sodium.

The lyophilized amorphous formulation that was produced resulted insurprising, unexpected and desirable results. The quantity ofundesirable and/or toxic degradation impurities that are produced fromthis formulation are less than those in current commercial products. Andthe stability of this formulation is superior to that of currentcommercial products. These results are particularly surprising andunexpected in view of the fact that conventional wisdom teaches thatcrystalline material is more stable than amorphous material (see e.g.,C. Ahlneck and G. Zografi “The Molecular Basis for Moisture Effects onthe Physical and Chemical Stability of Drugs in the Solid-State,” Int.J. Pharm. 1990, 62, 87-95). Thus, one of ordinary skill in the art wouldwant to avoid using amorphous material in pharmaceutical formulations.

Preferably the percentage of the lyophilized formulation that isamorphous is at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95%. Alternatively, the percentage of thelyophilized formulation that is amorphous can be 100%. In addition, thepercentage of the lyophilized formulation that is amorphous can bewithin any range between (and including) 20% to 100% (e.g., 20%-100%,25%-95%. etc.).

Methods for producing the storage-stable form of lyophilizedPhenobarbital Sodium composition result in a lyophilized powder in whichthe impurity phenylethylacetylurea (PEAU) is below detection limit andis never more than 0.2% of the specification limit even after storage ofthe lyophilized Phenobarbital Sodium for up to 6 months underaccelerated stability conditions (40° C., 75% RH). In some embodiments,the method for producing the storage-stable lyophilized PhenobarbitalSodium includes adding Phenobarbital Sodium to water to form aPhenobarbital Sodium solution, having a pH between 9.2 to 10.2, andlyophilizing the bulk solution. In some embodiments, the water is Waterfor Injection.

In preferred embodiments, the Water for Injection is sparged withnitrogen prior to adding the Phenobarbital Sodium. Typically, the Waterfor Injection is sparged with nitrogen for at least 30 minutes prior toadding the Phenobarbital Sodium. In additional embodiments, the water(e.g., Water for Injection) is cooled to or between 2 to 8° C. prior toadding the Phenobarbital Sodium.

For adjusting the pH of the Phenobarbital Sodium solution, the methodfor producing the storage-stable form of lyophilized PhenobarbitalSodium composition may also include measuring the pH of thePhenobarbital Sodium solution, wherein if the pH is higher than 10.2,the adjusting includes adding hydrochloric acid (HCl).

Typically, the resulting lyophilized Phenobarbital Sodium forms no morethan 0.1% PEAU when reconstituted in an aqueous solution after storageof the lyophilized Phenobarbital Sodium for up to 6 months both at RTand accelerated stability conditions. More typically, the resultinglyophilized Phenobarbital Sodium forms no more than 0.05% PEAU whenreconstituted in an aqueous solution after storage of the lyophilizedPhenobarbital Sodium for up to 6 months at room temperature (RT) andunder accelerated stability conditions (40° C.). Additionally, oralternatively, the resulting lyophilized Phenobarbital Sodium formseither an amount below the quantitative limit (BQL, 0.05%) or nodetectable amount of PEAU, 2-ethyl-2-phenylmalonamide (2EPMM), oralpha-phenylbutyrylguanidine (PBG) when reconstituted in the aqueoussolution after storage of the lyophilized Phenobarbital Sodium for up to6 months at RT and accelerated stability conditions.

According to contemplated embodiments as disclosed herein, the amorphouslyophilized Phenobarbital Sodium composition is stable up to at least 6months under accelerated stability conditions which is equivalent to 24months at RT. In this respect, in some embodiments, the stability of thelyophilized Phenobarbital Sodium is determined by reconstituting thelyophilized Phenobarbital Sodium in an aqueous solution and measuringthe amount of or the presence of phenylethylacetylurea (PEAU),2-ethyl-2-phenylmalonamide (2EPMM), and/or alpha-phenylbutyrylguanidine(PBG).

Additional embodiments include the method for producing thestorage-stable form of lyophilized Phenobarbital Sodium composition asdisclosed in which the Phenobarbital Sodium solution is filtered priorto lyophilization.

Further embodiments include the method for producing the storage-stableform of lyophilized Phenobarbital Sodium composition as disclosed inwhich the Phenobarbital Sodium solution is aliquoted into a vial priorto lyophilization, and after lyophilization, a closure is applied to thevial under vacuum.

Aspects of the present disclosure also include a method of reducingformation of degradation products of Phenobarbital Sodium reconstitutedin an aqueous solution, in which the method includes addingPhenobarbital Sodium to water to form a Phenobarbital Sodium solution,adjusting the pH of the Phenobarbital Sodium solution to or between 9.2to 10.2, and lyophilizing the Phenobarbital Sodium solution to formstable white lyophilized powder of Phenobarbital Sodium.

In preferred embodiments, the Water for Injection is sparged withnitrogen prior to adding the Phenobarbital Sodium. Typically, the Waterfor Injection is sparged with nitrogen for at least 30 minutes prior toadding the Phenobarbital Sodium. In additional embodiments, the water(e.g., Water for Injection) is cooled to or between 2 to 8° C. prior toadding the Phenobarbital Sodium.

For adjusting the pH of the Phenobarbital Sodium solution, the method ofreducing formation of degradation products of Phenobarbital Sodiumreconstituted in an aqueous solution may also include measuring the pHof the Phenobarbital Sodium solution, wherein if the pH is higher than10.2, the adjusting includes adding Hydrochloric acid (HCl).

In typical embodiments, the degradation products of Phenobarbital Sodiumreconstituted in an aqueous solution include phenylethylacetylurea(PEAU), 2-ethyl-2-phenylmalonamide (2EPMM), and/oralpha-phenylbutyrylguanidine (PBG), and analysis of the formation of thedegradation products includes reconstituting the stable lyophilizedPhenobarbital Sodium in an aqueous solution to form a reconstitutedsolution, and quantitating the amount of or presence of PEAU, 2 EPMM,and/or PBG in the reconstituted solution.

In typical embodiments, the method of reducing formation of degradationproducts of Phenobarbital Sodium reconstituted in an aqueous solutionresults in the reconstituted solution having no less than 96%Phenobarbital Sodium. In more typical embodiments, the reconstitutedsolution has no less than 97% Phenobarbital Sodium. In some preferredembodiments, the reconstituted solution has no less than 98%Phenobarbital Sodium, and in other preferred embodiments between 95% and105% Phenobarbital Sodium. In additional or alternative embodiments, thereconstituted solution has no more than 0.2% PEAU, 2 EPMM, and/or PBG,and preferably has 0.05% or less PEAU and no detectable amount of 2 EPMMor PBG.

Further aspects of the present disclosure include methods of treating anindividual in need of Phenobarbital Sodium. These methods include addingsaline or dextrose to the lyophilized powder of Phenobarbital Sodium asdisclosed herein immediately prior to administration to form aPhenobarbital Sodium reconstituted solution and administering thePhenobarbital Sodium reconstituted solution to the individual. In someembodiments, the individual in need of Phenobarbital Sodium suffers fromepilepsy. In particular embodiments, the individual in need ofPhenobarbital Sodium is a newborn suffering from neonatal epilepsy. Inother embodiments, the lyophilized amorphous Phenobarbital Sodium is apowder dose of 65 mg, 130 mg, 100 mg or 200 mg. In typical embodiments,the administering of the Phenobarbital Sodium reconstituted solution tothe individual includes intramuscular injection or intravenousinjection.

Additional aspects of the present disclosure include a pharmaceuticalproduct including a vial containing the white lyophilized powder ofPhenobarbital Sodium for reconstitution prior to administration, inwhich the composition was lyophilized in the vial and the vial includesa closure positioned in order to seal the vial under vacuum afterLyophilization.

Therefore, the inventors also contemplate lyophilized PhenobarbitalSodium.

Various objects, features, aspects, and advantages will become moreapparent from the following detailed description of preferredembodiments, along with the accompanying drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a polarized light photomicrograph of the presently disclosedPhenobarbital Sodium in water during Lyophilization microscopy at −50°C., as indicated.

FIG. 1B is a polarized light photomicrograph of the presently disclosedPhenobarbital Sodium in water during Lyophilization microscopy at −45.5°C. as indicated.

FIG. 1C is a polarized light photomicrograph of the presently disclosedPhenobarbital Sodium in water during Lyophilization microscopy at −40.0°C. as indicated.

FIG. 1D is a polarized light photomicrograph of the presently disclosedPhenobarbital Sodium in water during Lyophilization microscopy at −39.6°C. as indicated.

FIG. 1E is a polarized light photomicrograph of the presently disclosedPhenobarbital Sodium in water during Lyophilization microscopy at −39.2°C. as indicated.

FIG. 1F is a polarized light photomicrograph of the presently disclosedPhenobarbital Sodium in water during Lyophilization microscopy at −38.8°C. as indicated.

FIG. 2A is a chromatogram from an injection of a “blank solution” forassay by high performance liquid chromatography (HPLC) at a detectionwavelength of 210 nanometers (nm), as described herein.

FIG. 2B is a chromatogram from an injection of the “Sensitivitysolution” of Phenobarbital for related substances by HPLC at a detectionwavelength of 210 nm, as described herein.

FIG. 2C is a chromatogram from an injection of a “related substance”(RS) working standard at 0.2% impurity level) for assay of relatedsubstances by HPLC at a detection wavelength of 210 nm, as describedherein.

FIG. 2D is a chromatogram from an injection of2-ethyl-2-phenylmalonamide (2EPMM) for assay of related substances byHPLC at a detection wavelength of 210 nm, as described herein.

FIG. 2E is a chromatogram from an injection ofalpha-phenylbutyrylguanidine (PBG) for assay of related substances byHPLC at a detection wavelength of 210 nm, as described herein.

FIG. 2F is a chromatogram from an injection of phenylethylacetylurea(PEAU) for assay of related substances by HPLC at a detection wavelengthof 210 nm, as described herein.

FIG. 2G is a chromatogram from an injection of a “blank solution” forassay by HPLC at a detection wavelength of 254 nm, as described herein.

FIG. 2H is a chromatogram from an injection of an “assay workingstandard” for assay by HPLC at a detection wavelength of 254 nm, asdescribed herein.

FIG. 3A is a chromatogram from an injection of a marketed liquidformulation of Phenobarbital Sodium at 65 mg/mL at initial time pointfor assay by HPLC at a detection wavelength of 254 nm, as describedherein.

FIG. 3B is a chromatogram from an injection of a marketed liquidformulation of Phenobarbital Sodium at 65 mg/mL at 6 months from initialtime point at room temperature (RT) for assay by HPLC at a detectionwavelength of 254 nm, as described herein.

FIG. 3C is a chromatogram from an injection of a marketed liquidformulation of Phenobarbital Sodium at 65 mg/mL at 6 months from initialtime point at accelerated stability conditions (ACC) for assay by HPLCat a detection wavelength of 254 nm, as described herein.

FIG. 3D is a chromatogram from an injection of a marketed liquidformulation of Phenobarbital Sodium at 65 mg/mL at initial time pointfor related substances by HPLC at a detection wavelength of 210 nm, asdescribed herein.

FIG. 3E is a chromatogram from an injection of a marketed liquidformulation of Phenobarbital Sodium at 65 mg/mL at 6 months from initialtime point at room temperature (RT) for related substances by HPLC at adetection wavelength of 210 nm, as described herein.

FIG. 3F is a chromatogram from an injection of a marketed liquidformulation of Phenobarbital Sodium at 65 mg/mL at 6 months from initialtime point at accelerated stability conditions (ACC) for relatedsubstances by HPLC at a detection wavelength of 210 nm, as describedherein.

FIG. 3G is a chromatogram from an injection of a marketed liquidformulation of Phenobarbital Sodium at 130 mg/mL at initial time pointfor assay by HPLC at a detection wavelength of 254 nm, as describedherein.

FIG. 3H is a chromatogram from an injection of a marketed liquidformulation of Phenobarbital Sodium at 130 mg/mL at 6 months frominitial time point at room temperature (RT) for assay by HPLC at adetection wavelength of 254 nm, as described herein.

FIG. 3I is a chromatogram from an injection of a marketed liquidformulation of Phenobarbital Sodium at 130 mg/mL at 6 months frominitial time point at accelerated stability conditions (ACC) for assayby HPLC at a detection wavelength of 254 nm, as described herein.

FIG. 3J is a chromatogram from an injection of a marketed liquidformulation of Phenobarbital Sodium at 130 mg/mL at initial time pointfor related substances by HPLC at a detection wavelength of 210 nm, asdescribed herein.

FIG. 3K is a chromatogram from an injection of a marketed liquidformulation of Phenobarbital Sodium at 130 mg/mL at 6 months frominitial time point at room temperature (RT) for related substances byHPLC at a detection wavelength of 210 nm, as described herein.

FIG. 3L is a chromatogram from an injection of a marketed liquidformulation of Phenobarbital Sodium at 130 mg/mL at 6 months frominitial time point at accelerated stability conditions (ACC) for relatedsubstances by HPLC at a detection wavelength of 210 nm, as describedherein.

FIG. 4A is a chromatogram from an injection of the disclosedreconstituted lyophilized formulation of Phenobarbital Sodium at 65mg/mL at initial time point for assay by HPLC at a detection wavelengthof 254 nm, according to embodiments of the present invention.

FIG. 4B is a chromatogram from an injection of the disclosedreconstituted lyophilized formulation of Phenobarbital Sodium at 65mg/mL at 6 months from initial time point at room temperature (RT) forassay by HPLC at a detection wavelength of 254 nm, according toembodiments of the present invention.

FIG. 4C is a chromatogram from an injection of the disclosedreconstituted lyophilized formulation of Phenobarbital Sodium at 65mg/mL at 6 months from initial time point at accelerated stabilityconditions (ACC) for assay by HPLC at a detection wavelength of 254 nm,according to embodiments of the present invention.

FIG. 4D is a chromatogram from an injection of the disclosedreconstituted lyophilized formulation of Phenobarbital Sodium at 65mg/mL at initial time point for related substances by HPLC at adetection wavelength of 210 nm, according to embodiments of the presentinvention.

FIG. 4E is a chromatogram from an injection of the disclosedreconstituted lyophilized formulation of Phenobarbital Sodium at 65mg/mL at 6 months from initial time point at room temperature (RT) forrelated substances by HPLC at a detection wavelength of 210 nm,according to embodiments of the present invention.

FIG. 4F is a chromatogram from an injection of the disclosedreconstituted lyophilized formulation of Phenobarbital Sodium at 65mg/mL at 6 months from initial time point at accelerated stabilityconditions (ACC) for related substances by HPLC at a detectionwavelength of 210 nm, according to embodiments of the present invention.

FIG. 4G is a chromatogram from an injection of the disclosedreconstituted lyophilized formulation of Phenobarbital Sodium at 130mg/mL at initial time point for assay by HPLC at a detection wavelengthof 254 nm, according to embodiments of the present invention.

FIG. 4H is a chromatogram from an injection of the disclosedreconstituted lyophilized formulation of Phenobarbital Sodium at 130mg/mL at 6 months from initial time point at room temperature (RT) forassay by HPLC at a detection wavelength of 254 nm, according toembodiments of the present invention.

FIG. 4I is a chromatogram from an injection of the disclosedreconstituted lyophilized formulation of Phenobarbital Sodium at 130mg/mL at 6 months from initial time point at accelerated stabilityconditions (ACC) for assay by HPLC at a detection wavelength of 254 nm,according to embodiments of the present invention.

FIG. 4J is a chromatogram from an injection of the disclosedreconstituted lyophilized formulation of Phenobarbital Sodium at 130mg/mL at initial time point for related substances by HPLC at adetection wavelength of 210 nm, according to embodiments of the presentinvention.

FIG. 4K is a chromatogram from an injection of the disclosedreconstituted lyophilized formulation of Phenobarbital Sodium at 130mg/mL at 6 months from initial time point at room temperature (RT) forrelated substances by HPLC at a detection wavelength of 210 nm,according to embodiments of the present invention.

FIG. 4L is a chromatogram from an injection of the disclosedreconstituted lyophilized formulation of Phenobarbital Sodium at 130mg/mL at 6 months from initial time point at accelerated stabilityconditions (ACC) for related substances by HPLC at a detectionwavelength of 210 nm, according to embodiments of the present invention.

FIG. 5A is an X-ray powder diffraction (XRPD) pattern of PhenobarbitalSodium Grade P

FIG. 5B is an XRPD pattern of Phenobarbital Sodium for Injection, USP,65 mg/vial.

FIG. 5C is an XRPD pattern of Phenobarbital Sodium for Injection, USP,130 mg/vial.

FIG. 5D is a stack plot of the XRPD patterns of FIGS. 5A, 5B, and 5C.

DETAILED DESCRIPTION

The inventors have surprisingly discovered a method for making astorage-stable and highly purified Phenobarbital Sodium composition thatis void of any detectable impurities in its lyophilized powder form andis essentially void of impurities upon reconstitution in water. Thecontemplated Phenobarbital Sodium composition is a lyophilized powderthat is no less than 98% Phenobarbital Sodium and is stable (25° C., 65%RH and 40° C., 75% RH) for up to at least 6 months under acceleratedconditions which is equivalent to 24 months at RT. Upon reconstitution(e.g. solubilization/resuspension) in water, the lyophilizedPhenobarbital Sodium forms no more than 0.05% of2-ethyl-2-phenylmalonamide (2EPMM) or alpha-phenylbutyrylguanidine(PBG), and phenylethylacetylurea (PEAU).

The Phenobarbital Sodium composition as described herein refers toPhenobarbital Sodium having an IUPAC (International Union of Pure andApplied Chemistry) name of sodium5-ethyl-4,6-dioxo-5-phenyl-1H-pyrimidin-2-olate, a Chemical AbstractService (CAS) No. 57-30-7, and a molecular formula of C₁₂H₁₁N₂NaO₃.

In order to avoid hydrolysis of Phenobarbital Sodium and therefore theformation of its toxic degradation products, the inventors haveunexpectedly discovered that a lyophilized Phenobarbital Sodium formedby the methods disclosed herein results in a storage-stable lyophilizedpowder of Phenobarbital Sodium. In particular, with reference to thepolarized light photomicrographs of FIGS. 1A-1F and the XRPD analysisshown in FIG. 5A-5D, the storage-stable lyophilized powder ofPhenobarbital Sodium has an amorphous structure. Without wishing to bebound by any theory or hypothesis, the lyophilized Phenobarbital Sodiumproduced following the methods according to embodiments of the presentdisclosure produces an amorphous structure that contributes to theobserved stability of this composition upon reconstitution in water.Irrespective of the mechanism of action, the contemplated lyophilizedPhenobarbital Sodium composition is stable and upon reconstitution inwater, does not form significant (e.g., not measurable or notdetectable) amounts of the toxic degradation products—namely PEAU,2EPMM, or PBG.

The contemplated method for producing a storage-stable lyophilizedPhenobarbital Sodium composition includes adding purified PhenobarbitalSodium to water. Preferably the purified Phenobarbital Sodium solid ispurchased from a chemical supplier that adheres to the United StatesPharmacopeia (USP) manufacturing and synthesis guidelines. The USP gradeof the Phenobarbital Sodium is preferably USP “purified” or “practical”(P) grade indicating that it is a good quality chemical. As understoodin the art, the USP grade (P) active pharmaceutical ingredient (API) ofPhenobarbital Sodium is not suitable for administration because it isnot sterile, is hygroscopic, and as such, maintaining anhydrousconditions of this grade of API is not feasible. Structurally, theanalyzed USP grade P API is polymorphous—containing both crystalline andnon-crystalline material. With reference to FIG. 5A, an X-ray powderdiffraction (XRPD) pattern is shown of this compound.

Considering the need to avoid hydrolysis of the Phenobarbital Sodium,the Phenobarbital Sodium is preferably added to Water for Injection andlater lyophilized to obtain a stable lyophilized finished product. Inorder to remove dissolved oxygen (O₂) gas from the water (e.g., Waterfor Injection), it is also preferred that the water is sparged withnitrogen (N₂) or other inert gas prior to the addition of thePhenobarbital Sodium solid. For effective sparging, a volume of about100-200 ml water is sparged with nitrogen gas for at least 30 minutes.In exemplary protocols for producing either 65 mg, 130 mg of lyophilizedPhenobarbital Sodium, 100 milliliters (ml) of Water for Injection isnitrogen sparged for 30 minutes. In additional embodiments, the watermay also be cooled to approximately 2 to 8° C. In preferred embodiments,the water is cooled to approximately 2 to 8° C.

The addition of the Phenobarbital Sodium solid to the water (e.g.,deionized nitrogen sparged water cooled to 2 to 8° C.) may be carriedout using any suitable mixing. Preferably, the water (e.g., Water forInjection) is first added to a glass/stainless steel container fornitrogen sparging and cooling, and this prepped water remains in theglass/stainless steel container or a measured amount is transferred to apreferably weighed glass container/stainless steel container and thePhenobarbital Sodium solid is added thereto. For mixing, any suitablemixing may be used. For example, a magnetic stir bar may be added to theglass/stainless steel container. Upon addition of the PhenobarbitalSodium solid to the water, the mixture is stirred.

An additional consideration for stability of the Phenobarbital Sodium ismaintaining a pH between 9.2 and 10.2 in the bulk solution ofPhenobarbital Sodium. After dissolution of the Phenobarbital Sodiumsolid in the water, the pH is adjusted to within this range of 9.2 and10.2. Accordingly, the pH of the water solubilized Phenobarbital Sodiumis first measured upon dissolution. If the pH is higher than 10.2 orlower than 9.2, the pH is adjusted. For example, if necessary, the pHmay be lowered to or between 9.2 and 10.2 using Hydrochloric acid (HCl)and the pH may be increased to or between 9.2 and 10.2 using SodiumHydroxide (NaOH).

Following the pH measurement and any adjustment, the PhenobarbitalSodium solution is preferably filtered to remove any foreign particles.For example, the Phenobarbital Sodium solution may be filtered using a0.2 μm (or 0.22 μm) membrane filter. For example, the membrane filtermay be polyvinylidene difluoride (PVDF) or any other compatible filtermembranes such as Polyethersulfone (PES) filter.

According to aspects of the contemplated method, the PhenobarbitalSodium solution dissolved in the water (e.g., nitrogen sparged Water forInjection cooled to 2 to 8° C.) having a pH of or between 9.2 and 10.2and preferably filtered is ready for Lyophilization. Depending on thedesired amount of the lyophilized Phenobarbital Sodium product, thePhenobarbital Sodium solution may be lyophilized in any amount or thesolution may be aliquoted into sterile containers (e.g., vials). Forexample, in order to obtain a measured dose of lyophilized PhenobarbitalSodium in a container, the glass container, water, and PhenobarbitalSodium solid are weighed prior to mixing in order to determine an actualweight of the Phenobarbital Sodium after pH measurement and adjustment.For example, to produce 65 mg or 130 mg doses of lyophilizedPhenobarbital Sodium, a batch may be dissolved, mixed, pH adjusted ifnecessary, and filtered and then aliquoted into single use dose vialsfor lyophilization. In a specific example, a 50 mL batch ofPhenobarbital Sodium solution may be prepared where for 65 mg doses,3.25 grams of Phenobarbital Sodium solid (e.g., USP Grade P) is added to40 ml of water (e.g., nitrogen sparged Water for Injection cooled to 5to 8° C.) and for 130 mg doses, 6.5 grams of the Phenobarbital Sodium isadded to the 40 ml of water. Following dissolution, pH adjustment ifnecessary, adjusting final volume to 50.0 mL and filtering, 1 mlaliquots of this Phenobarbital Sodium solution are added to open (e.g.,unstoppered) vials and then loosely stoppered (e.g., with lyophilizationrubber stoppers) prior to lyophilization. Following lyophilization, eachvial contains 65 mg or 130 mg of lyophilized Phenobarbital Sodium.

In further embodiments, following lyophilization, the vials may becompletely closed (e.g., stoppered) under vacuum.

Another embodiment is directed to a pharmaceutical composition thatconsists of lyophilized amorphous Phenobarbital Sodium.

Another embodiment is directed to a method of producing a storage-stableform of lyophilized Phenobarbital sodium that forms no more than 0.2%phenylethylacetylurea (PEAU) when reconstituted in an aqueous solution.The method comprising: includes adding Phenobarbital Sodium to water toform a Phenobarbital Sodium solution, if necessary, adjusting the pH ofthe Phenobarbital Sodium solution to or between 9.2 to 10.2; andlyophilizing the Phenobarbital Sodium solution.

In one embodiment, the water is Water for Injection.

In another embodiment the Water for Injection is sparged with nitrogenprior to adding the Phenobarbital Sodium.

In another embodiment, the Water for Injection is sparged with nitrogenfor at least 30 minutes prior to adding the Phenobarbital Sodium.

In another embodiment, the water is cooled to or between 2 to 8° C.prior to adding the Phenobarbital Sodium.

In another embodiment, the adjusting includes measuring the pH of thePhenobarbital Sodium solution, and if the pH is higher than 10.2, theadjusting includes adding Hydrochloric acid (HCl).

In another embodiment, the lyophilized Phenobarbital Sodium forms nomore than 0.1% phenylethylacetylurea (PEAU) when reconstituted in anaqueous solution after storage of the lyophilized Phenobarbital Sodiumfor up to 6 months at room temperature (RT) and accelerated stabilityconditions (ACC).

In another embodiment, the lyophilized Phenobarbital Sodium forms nomore than 0.05% phenylethylacetylurea (PEAU) when reconstituted in anaqueous solution after storage of the lyophilized Phenobarbital Sodiumfor up to 6 months at room temperature (RT) and accelerated stabilityconditions (ACC).

In another embodiment, the lyophilized Phenobarbital Sodium forms nodetectable amount of 2-ethyl-2-phenylmalonamide (2EPMM) oralpha-phenylbutyrylguanidine (PBG) when reconstituted in an aqueoussolution after storage of the lyophilized Phenobarbital Sodium for up to6 months at room temperature (RT) and accelerated stability conditions(ACC).

In another embodiment, the lyophilized Phenobarbital Sodium is stable upto at least 6 months at accelerated stability conditions (ACC) which isequivalent to 24 months at room temperature (RT).

In another embodiment, the stability of the lyophilized PhenobarbitalSodium is determined by adding the lyophilized Phenobarbital Sodium toan aqueous solution to form a reconstituted lyophilized PhenobarbitalSodium and analyzing to quantitate the amount of phenylethylacetylurea(PEAU), 2-ethyl-2-phenylmalonamide (2EPMM), and/oralpha-phenylbutyrylguanidine (PBG) in the reconstituted lyophilizePhenobarbital Sodium.

In another embodiment, the Phenobarbital Sodium solution is at aconcentration of 65 mg/mL, 100 mg/mL, 130 mg/mL or 200 mg/mL.

In another embodiment, the method further includes filtering thePhenobarbital Sodium solution prior to lyophilizing.

In another embodiment, the method further includes aliquoting thePhenobarbital Sodium solution into a vial prior to lyophilization; andafter lyophilization, applying a closure to the vial under vacuum.

Another embodiment is directed to a method of treating an individual inneed of Phenobarbital Sodium. The method includes adding saline ordextrose to a composition containing lyophilized amorphous PhenobarbitalSodium immediately prior to administration to form a PhenobarbitalSodium solution; and administering the Phenobarbital Sodium solution tothe individual.

In another embodiment, the individual suffers from epilepsy.

In another embodiment, the individual is a newborn suffering fromneonatal epilepsy.

In another embodiment, the composition is a powder dose of 65 mg, 100,130 mg or 200 mg.

In another embodiment, the administering includes intramuscularinjection or intravenous injection.

Surprisingly, the inventors have determined that the presently disclosedmethod produces a storage-stable lyophilized Phenobarbital Sodium thatmaintains its stability up to at least 6 months under acceleratedstability conditions which is equivalent to 24 months at RT. Withreference to FIGS. 5B-5C, the lyophilized Phenobarbital Sodium isamorphous. In particular, the lyophilized Phenobarbital Sodium isnon-crystalline unlike the USP grade P API shown comparatively in FIG.5D. The lyophilized Phenobarbital Sodium is stable and ready forreconstitution in either water for injection, 0.9% Saline or 5% Dextrosefor intravenous (IV) or intramuscular (IM) administration. The presentlydisclosed formulation does not contain any organic solvents orpreservatives which is specifically advantageous for administration tonewborns and neonates. Moreover, the resulting lyophilized PhenobarbitalSodium forms below the quantitation limit of 0.05% phenylethylacetylurea(PEAU), and no detectable amount of 2-ethyl-2-phenylmalonamide (2EPMM)or alpha-phenylbutyrylguanidine (PBG) when reconstituted in water afterstorage of the lyophilized Phenobarbital Sodium for up to 6 months underaccelerated stability conditions which is equivalent to 24 months at RT.

In a comparison analysis, the presently disclosed lyophilized powder ofPhenobarbital Sodium at 65 mg or 130 mg doses was reconstituted in waterand assayed for impurities and stability along with marketed liquidformulation of Phenobarbital Sodium for Injection USP, 65 mg/mL or 130mg/mL (Westward Pharmaceuticals). The formulation details for thiscomparative analysis are presented in Table 1. Representativechromatograms from HPLC analysis are shown in FIGS. 2A-2H, 3A-3L, and4A-4L.

TABLE 1 Formulation Details Dosage form Liquid Lyophilized ComponentsChemical name mg/mL mg/vial Drug Phenobarbital 65 or 130 65 or 130Substance Sodium Inactive Propylene 705 NA ingredients Glycol Benzylalcohol 15.6 NA Ethanol 78.9 NA HCL For pH adjustment NA Water for QS NAInjection (Quantity Supplied)

With reference to Tables 2 and 3, the presently disclosed 65 mglyophilized Phenobarbital Sodium composition was reconstituted in waterand compared to the 65 mg Liquid Phenobarbital Sodium for Injection USP(65 mg/mL marketed Liquid Formulation) (Table 2) (FIGS. 3A-3F, FIGS.4A-4F), and the presently disclosed 130 mg lyophilized PhenobarbitalSodium for Injection USP (130 mg/vial) composition was reconstituted inwater and compared to the 130 mg liquid Phenobarbital Sodium forInjection USP (130 mg/mL marketed Liquid Formulation) (Table 3) (FIGS.3G-3L, FIGS. 4G-4L). In addition to the noted characteristics of eachproduct, the products were assayed for the degradationproducts—phenylethylacetylurea (PEAU), 2-ethyl-2-phenylmalonamide(2EPMM), and alpha-phenylbutyrylguanidine (PBG) by HPLC analysis, asfurther described in the Examples. As indicated in both Tables 2 and 3,the initial amount of PEAU (phenylethylacetylurea) assayed in the 65mg/mL and 130 mg/mL liquid formulations was 0.5% and 0.5%, respectively.Comparatively, no initial amount of PEAU was detected in either the 65mg or 130 mg lyophilized Phenobarbital Sodium compositions. Furthermore,after 3 and 6 months from the date of manufacture, both the liquid andlyophilized formulations at 65 mg/mL and 130 mg/mL stored at roomtemperature (RT) (25° C.) and under accelerated stability conditions(ACC) at 40° C. were analyzed by HPLC for assay and related substances.Under these conditions, the amount of PEAU assayed in the 65 mg/mLliquid formulations at 3 months was 1.0% (RT) and 3.7% (ACC) and at 6months was 1.4% (RT) and 5.8% (ACC) (Table 2). Similarly, the amount ofPEAU assayed in 130 mg/mL liquid formulations at 3 months was 0.9% (RT)and 2.8% (ACC) and at 6 months was 1.1% (RT) and 4.3% (ACC) (Table 3).In contrast, for the presently disclosed lyophilized compositions, theamount of PEAU assayed at 3 and 6 months for the 65 mg and the 130 mglyophilized Phenobarbital Sodium compositions was below quantitationlimit (BQL) (0.05%) both at RT and ACC stability conditions (Tables2-3).

TABLE 2 65 mg/ml Liquid Formulation and 65 mg/vial Lyophilized PowderProduct Name Phenobarbital Sodium for Injection USP, PhenobarbitalSodium for Injection USP, 65 mg/mL (Marketed product) 65 mg/Vial(Nivagen formulation- NPH1907) Formulation Type Liquid Lyophilizedpowder Stability Data 3 Months 6 Months 3 Months 6 Months 15 Months RTACC RT ACC RT ACC RT ACC RT** 25° C., 40° C., 25° C., 40° C., 25° C.,40° C., 25° C., 40° C., 25° C., 60% 75% 60% 75% 60% 75% 60% 75% 60% TestInitial RH RH RH RH Initial RH RH RH RH RH Description CCVS CCVS CCVSCCVS CCVS WLP WLP WLP WLP WLP WLP Appearance NA NA NA NA NA CCS CCS CCSCCS CCS CCS after reconstitution Identification $ $ $ $ $ $ $ $ $ $ $pH* 9.3 9.8 9.7 9.6 9.6 9.8 9.6 9.6 9.7 9.7 9.8 % Assay of 102.4  97.6 103.1  96.3  86.7  99.1  98.7  97.4  100.0 99.3 96.0 PhenobarbitalSodium % degradation ND ND BQL BQL BQL ND ND ND ND ND ND (Area percent)2EPMM PBG ND ND ND ND ND ND ND ND ND ND ND PEAU 0.5 1.0 3.7 1.4 5.8 NDBQL BQL BQL BQL BQL Any BQL BQL 0.1 BQL 0.3 BQL BQL BQL 0.07 0.06 0.04unspecified degradation product Total 0.5 1.0 3.8 1.4 6.1 BQL BQL BQL0.07 0.06 0.04 degradation products CCVS: Clear Colorless ViscousSolution; CCS: Clear Colorless Solution; WLP: White Lyophilized powder$: In Assay analysis, the retention time of Phenobarbital in standardand sample solutions should be same. BQL: Below Quantitation Limit;(QL-0.05%); ND: Not Detected 2EPMM: 2-Ethyl-2-Phenylmalonamide,monohydrate; PBG: α-phenylbutyrylguanidine, PEAU: phenylethylacetylureaor Pheneturide *pH of both liquid and lyophilized formulations aremeasured at 65 mg/mL concentration **The samples were stored at 30 ± 2°C., 65% RH ± 5 Notably, the Lyophilized powder was stable even after 15months, and no significant increase in the impurities was observed,rendering such formulation superior to known liquid formulations andenable easy administration.

TABLE 3 130 mg/mL Liquid Formulation and 130 mg/vial Lyophilizedformulation Product Name Phenobarbital Sodium for Injection USP,Phenobarbital Sodium for Injection USP, 130 mg/mL (Marketed product) 130mg/vial (Nivagen formulation-NPH1912) Formulation Type LiquidLyophilized powder Stability Data 3 Months 6 Months 3 Months 6 Months 15Months RT ACC RT ACC RT ACC RT ACC RT** 25° C., 40° C., 25° C., 40° C.,25° C., 40° C., 25° C., 40° C., 25° C., 60% 75% 60% 75% 60% 75% 60% 75%60% Test Initial RH RH RH RH Initial RH RH RH RH RH Description CCVSCCVS CCVS CCVS CCVS WLP WLP WLP WLP WLP WLP Appearance NA NA NA NA NACCS CCS CCS CCS CCS CCS after reconstitution Identification $ $ $ $ $ $$ $ $ $ $ pH* 9.5 9.9 9.9 9.8 9.7 9.8 9.8 9.8 9.8 9.8  10.0  % Assay of100.8  95.5  92.9  95.2  90.9  98.3  100.1  97.9  100.2 99.4  98.3 Phenobarbital Sodium Related ND ND BQL BQL BQL ND ND ND ND ND NDSubstances (% Area) 2EPMM PBG ND ND ND ND ND ND ND ND ND ND ND PEAU 0.50.9 2.8 1.1 4.3 ND BQL BQL BQL BQL BQL Any ND BQL 0.1 BQL 0.4 BQL BQLBQL 0.05 0.05 0.04 unspecified degradation product Total 0.5 0.9 2.9 1.14.7 BQL BQL BQL 0.05 0.05 0.04 degradation products CCVS: ClearColorless Viscous Solution; CCS: Clear Colorless Solution; WLP: WhiteLyophilized powder $: In Assay analysis, the retention time ofPhenobarbital in standard and sample solutions should be same. BQL:Below Quantitation Limit; (QL-0.05%); ND: Not Detected 2EPMM:2-Ethyl-2-Phenylmalonamide, monohydrate; PBG: α-phenylbutyrylguanidine,PEAU: phenylethylacetylurea or Pheneturide *pH for liquid formulation ismeasured at 130 mg/mL and for lyophilized formulation at 100 mg/mLdilution **The samples were stored at 30 ± 2° C., 65% RH ± 5

In typical embodiments, the method of reducing formation of degradationproducts of a lyophilized Phenobarbital Sodium composition reconstitutedin water results in the reconstituted solution having no less than 96%Phenobarbital Sodium. In more typical embodiments, the reconstitutedsolution has no less than 97% Phenobarbital Sodium. In more preferredembodiments, the reconstituted solution has no less than 98%Phenobarbital Sodium. In additional or alternative embodiments, thereconstituted solution has no quantitative amount (e.g., 0.05% or less)of PEAU, 2 EPMM, or PBG.

According to contemplated embodiments, assaying the stability of thelyophilized Phenobarbital Sodium composition includes reconstituting thecomposition in water at a concentration of 65 mg/mL, 100 mg/mL, 130mg/mL or 200 mg/mL.

Further aspects of the present disclosure include methods of treating anindividual in need of Phenobarbital Sodium. For administration to anindividual (e.g., a human patient) the lyophilized powder ofPhenobarbital Sodium powder may be reconstituted in saline or dextroseas disclosed herein immediately prior to administration to form aPhenobarbital Sodium reconstituted solution. In particular embodiments,the individual in need of Phenobarbital Sodium suffers from epilepsy. Inpreferred embodiments, the individual in need of Phenobarbital Sodium isa newborn suffering from neonatal epilepsy. In other embodiments, thelyophilized Phenobarbital Sodium is a powder dose of 65 mg, 100 mg, 130mg or 200 mg. In typical embodiments, the administering of thePhenobarbital Sodium reconstituted solution to the individual includesintramuscular injection or intravenous injection.

EXAMPLES

Lyophilization Microscopy. Lyophilization microscopy (FIGS. 1A-1F) wasperformed using a Leica DM LP microscope equipped with a Linkam FDCS196stage, TMS93 controller, vacuum pump, LNP unit, and a Spot Insight colorcamera. A 20×, 040 N.A. objective was used with crossed polarizers and afirst order red compensator to view sample. Images were acquired usingSpot Advanced software (v.4.5.9).

Lyophilization parameters for the lyophilization microscopy including astart temperature of −50° C. with heating at 1° C./minute to −40° C.followed by heating at 0.5° C./minute to −32.0° C.

HPLC Assays. High Performance Liquid Chromatography (HPLC) was used toassay Phenobarbital Sodium standards and sample solutions as disclosedherein (e.g., Tables 2, 3, FIGS. 2A-2H, 3A-3L, and 4A-4L). Both UltraHigh-Performance Liquid Chromatographic system (UHPLC) andHigh-Performance Liquid Chromatographic system (HPLC)(ThermoFisher/Waters) were used together with Data Acquisition Software(Empower 3), equipped with UV and Photodiode Array Detector (UV andPDA), column thermostat and auto sampler compartments or equivalent HPLCsystem and software.

An exemplary HPLC assay protocol is provided in the following:

Instruments/Apparatus: HPLC with UV detector/PDA Detector, Analyticalbalance, Sonicator, Volumetric flasks, Beakers, Pipettes.

Reagents, Solvents, Standards: Phenobarbital Reference Standard,Deionized water/Water for Injection, Potassium Monobasic Phosphate,Acetonitrile.

Preparation of Mobile Phase Solutions: Potassium phosphate monobasic (7mM) and Acetonitrile (70:30) pH 4.5-6.5; Diluent: Mobile phase as adiluent.

Wash Solvent: Water: Acetonitrile (70:30)

CHROMATOGRAPHIC CONDITIONS: The liquid chromatography equipped with a

Parameters Conditions Column L7 packaging column, 150 × 4.6 mm, ParticleSize: 5 μm or equivalent Flow rate 0.9-1.2 mL/min. Detection Wavelength254 ± 2 nm for assay and 210 ± 2 nm for Related Substances Injectionvolume 10 μL Run time 15 Minutes Column Oven 30° C. Temperature SamplerCooler 25° C. Temperature Retention Time About 5 minutes forPhenobarbital Pump Mode Isocratic UV and PDA detector, an injector and adata processor is operated as follows:

Phenobarbital Assay Standard Solution: 0.2 mg/mL to 0.8 mg/mL indiluent; Phenobarbital Related Substances Standard Solution: 0.26 μg/mLto 15.6 μg/mL in diluent; Phenobarbital Related Substances resolutionstandard solution: 0.475 mg/mL Phenobarbital and about 1μg/mL forrelated substances (2-Ethyl-2-Phenyl malonamide, monohydrate (2EPMM),Phenylbutyrylguanidine (PBG) and Phenylethylacetylurea (PEAU) indiluent; Phenobarbital Sodium Sample Solution for Assay: 0.26 mg/mL to0.78 mg/mL in diluent

Phenobarbital Sodium related Substances sample solution: 0.52 mg/mL ofPhenobarbital Sodium and related substances 2-Ethyl-2-Phenylmalonamide,monohydrate (2EPMM), Phenyl butyrylguanidine (PBG) andPhenylethylacetylurea (PEAU) and any unspecified degradation products at0.26 μg/mL to 13.2 μg/mL

SYSTEM SUITABILITY REQUIREMENTS: The relative standard deviation ofPhenobarbital peak from Six replication injections of related substancesstandard should not be more than 6.0%. The related standard deviation ofPhenobarbital peak from five replicate injections in assay standardsolution should not be more than 2.0%. Tailing factor for thePhenobarbital is not more than 2.0. USP Plate count for thePhenobarbital is not less than 2500. The S/N Ratio of the SensitivityStandard Solution is NLT 10 for related substance test. The resolutionbetween Phenobarbital peak and nearest impurity peak is NLT 1.5

Two alternative formulations are disclosed in Table 6. One of theseformulations is 200 mg/vial, Phenobarbital Sodium reconstituted in 10 mLof Water for Injection or 5% Dextrose to obtain a concentration of 20mg/mL. A second formulation is 200 mg/vial of Phenobarbital Sodium alongwith 90 mg Sodium Chloride reconstituted in 10 mL of Water for Injectionto obtain a concentration of 20 mg/mL Phenobarbital Sodium for Injectionand 9 mg/mL or 0.9% Sodium Chloride.

TABLE 6 Phenobarbital Sodium for Injection, USP, 200 mg/vial in 10 mLvial. Formulation Details. NPH2031 A NPH2031 A (Bulk Solution at (BulkSolution at 100 mg · mL 100 mg · mL Phenobarbital Phenobarbital Sodiumand 45 mg/mL Sodium) Sodium Chloride) Compounding at 2-8° C. Compoundingat 2-8° C. Fill Volume: 2.1 mL mg/mL (after mg/mL (after Ingredientsreconstitution) mg/vial reconstitution) mg/vial Phenobarbital 20 200 20200 Sodium Sodium NA NA 9 90 Chloride WFI/DI qs qs qs qs WaterReconstitution Volume 10 mL; Diluents that can be used are Water forInjection, 0.9% Saline or 5% Dextrose.

These formulations along with the previously disclosed formulations weredeveloped to enable physicians and medical staff at hospitals to easilyreconstitute to a desired concentration (e.g., 20 mg/mL) and administerthe medicine to patients in need thereof. The reconstituted solution isstable at 2-8° C. for 24 hrs and 3 to 4 hrs when stored at RT (see e.g.,Tables 7, 8 and 9).

TABLE 7 Reconstituted Solution Stability of Phenobarbital Sodium forInjection USP, 200 mg/vial (NPH2031A Phenobarbital Sodium @ 200 mg/vial)(Reconstitution in 10 mL Water) Initial Reconstituted Solution Stabilityafter 20 hrs DOA: DOA: Jun. 6, 2020 Test Spec. Jun. 5, 2020 2-8° C. RTDescription WLP WLP WLP WLP Appearance CCS CCS CCS CCS pH (20 9.2-10.29.9 9.7  9.8  mg/mL) % Assay 90.0-105.0 100.1  100.1   100.1  Phenobarbital Sodium Related Substances (% Area) NMT 0.2% BQL ND ND2EPMM PBG NMT 0.2% ND ND ND PEAU NMT 0.2% BQL 0.03 0.03 Any unspecifiedNMT 0.2% BQL 0.06 0.24 degradation product Total degradation productsNMT 1.5% BQL 0.09 0.27 Water Content by Karl Fischer NMT 3.0%  0.84 NANA Note Book Reference 192045-163 to 192045- 163-183 189 DOA: Date ofAnalysis; WLP: White Lyophilized Powder; CCS: Clear Colorless Solution,1 minute after reconstitution; ND: Not Detected; NMT: Not More Than;BQL: Below Quantitation Limit (0.05%) 2EPMM: 2-Ethyl-2-Phenylmalonamide,monohydrate; PBG: α-Phenylbutyrylguanidine; PEAU: Phenylethylacetylurea

TABLE 8 Reconstituted Solution Stability of Phenobarbital Sodium forInjection USP, 200 mg/vial in Sodium Chloride (NPH2031B PhenobarbitalSodium @ 200 mg/vial and Sodium Chloride @ 90 mg/vial) (Reconstitutionin 10 mL Water) Reconstituted Solution Stability after 20 hrs TestSpecification Initial 2-8° C. RT Description WLP WLP WLP WLP AppearanceCCS CCS CCS CCS pH (20 mg/mL) 9.2-10.2 9.8 9.7  9.8 % Assay 90.0-105.0103.3 103.4   102.8 Phenobarbital Sodium Related NMT 0.2% BQL ND BQLSubstances (% Area) 2EPMM PBG NMT 0.2% ND ND ND PEAU NMT 0.2% BQL 0.030.03 Any NMT 0.2% BQL 0.06 0.27 unspecified degradation product TotalNMT 1.5% BQL 0.09 0.3 degradation products Water Content NMT 3.0% 0.84NA NA by Karl Fischer WLP: White Lyophilized Powder; CCS: ClearColorless Solution, 1 minute after reconstitution; ND: Not Detected;NMT: Not More Than; BQL: Below Quantitation Limit (0.05%) 2EPMM:2-Ethyl-2-Phenylmalonamide, monohydrate; PBG: α-Phenylbutyrylguanidine;PEAU: Phenylethylacetylurea

TABLE 9 Reconstituted Solution Stability of Phenobarbital Sodium forInjection USP, 130 mg/vial (NPH2030) (20 mg/mL dilution In Water) TimeShelf life 2 hrs 4 hrs 24 hrs Tests Specifications Initial 2-8 C. RT 2-8C. RT 2-8 C. RT Appearance CCS CCS CCS CCS CCS CCS CCS CCS pH (20 mg/mL9.2-10.2 9.5 9.5 9.6 9.6 9.5 9.6 9.5 dilution) Assay of 90.0%-105.0%99.8 102.1 100.8 101.7 101.2 100.4 100.3 Phenobarbital Sodium RelatedSubstances NMT 0.2% ND ND ND ND ND ND BQL (% Area) 2EPMM PBG NMT 0.2% NDND ND ND ND ND ND PEAU NMT 0.2% BQL BQL BQL BQL BQL BQL BQL Anyunspecified NMT 0.2% 0.015 0.02 0.04 0.02 0.06 0.04 0.18 degradationproduct @ RRT 0.37 Total NMT 1.5% 0.015 0.02 0.04 0.02 0.06 0.04 0.18degradation products Osmolality To be reported 158 NP NP NP NP 159 158mOsmol/Kg CCS: Clear colourless solution; ND: Not detected; BQL: BelowQuantitation limit, NP: Not performed; 2EPMM: 2-ethylphenylmalonamidemonohydrate; PBG: Phenylbutyrylguanidine; PEAU: Phenylethylacetylurea

Six months stability data for Phenobarbital Sodium for Injection, USP,65 mg/vial is included in Table 10. Six months stability data forPhenobarbital Sodium for Injection, USP, 130 mg/vial is included inTable 11. Three months stability data for Phenobarbital Sodium forInjection, USP, 200 mg/vial is included in Table 12. Three monthsstability data for Phenobarbital Sodium for Injection, USP(Phenobarbital Sodium and 9% Saline), 200 mg/vial is included in Table13.

TABLE 10 Product Name Phenobarbital Sodium for Injection USP, 65 mg/vialBatch#NPH2020 Fill Volume: 1.05 mL Date Manufactured: Jan. 20, 2020 VialMake 2 mL Clear Glass Vial: 13 mm Lyo rubber stopper; Seal: 13 mm Flipoff plastic with Al seal. API: Phenobarbital Sodium, USP Grade PConfiguration Inverted Stability 25° C., 60% RH (RT) & 40° C., 75% RH(ACC) Initial 1 Month 3 Months 6 Months DOA: DOA: Mar. 23, 2020 DOA: May20, 2020 DOA: Sep. 4, 2020 Test Spec. Feb. 18, 2020 RT ACC RT ACC RT ACCDescription WLP WLP WLP WLP WLP WLP WLP WLP Appearance CCS CCS CCS CCSCCS CCS CCS CCS pH (65 mg/mL) 9.2-10.2 9.9 9.9 9.9 9.9 9.9 9.6 9.6 %Assay 90.0%-105.0% 98.6  101.6  101.5  101.3  100.5  100.0  101.2 Phenobarbital Sodium Related NMT 0.2% BQL ND ND ND ND ND ND Substances(% Area) 2EPMM PBG NMT 0.2% ND ND ND ND ND ND ND PEAU NMT 0.2% BQL BQLBQL BQL BQL BQL BQL Any NMT 0.2% BQL BQL BQL BQL BQL BQL BQL unspecifieddegradation product Total NMT 1.5% BQL BQL BQL BQL BQL BQL BQLdegradation products Water Content NMT 3.0% 0.6 0.9 1.3 1.4 1.5 1.5 2.1by Karl Fischer Note Book Reference 192039-150 to 158 192045- 23 to 45192045-135 to 153 192051-84 to 122 DOA: Date of Analysis; WLP: WhiteLyophilized Powder; CCS: Clear Colorless Solution, 1 minute afterreconstitution; ND: Not Detected; NMT: Not More Than; BQL: BelowQuantitation Limit (0.05%) 2EPMM: 2-Ethyl-2-Phenylmalonamide,monohydrate; PBG: α-Phenylbutyrylguanidine; PEAU: Phenylethylacetylurea;

TABLE 11 Product Name Phenobarbital Sodium for Injection USP, 130mg/vial Batch#NPH2021 Fill Volume: 1.05 mL Date Manufactured: Jan. 20,2020 Vial Make 2 mL Clear Glass Vial Date Initiated: Feb. 14, 2020 APIMfg. Phenobarbital Sodium, USP Grade P, Re test Date: Nov. 16, 2023Stopper Used 13 mm Grey Rubber stopper Flip Off Seals: 13 mm Plasticwith Aluminum Seal Configuration Inverted Stability 25° C., 60% RH (RT)& 40° C., 75% RH (ACC) 1 Month 3 Months 6 Months 24 Months Initial DOA:DOA: DOA: DOA: DOA: Mar. 23, 2020 May 20, 2020 Sep. 4, 2020 18 Mar. 2022Test Spec. Feb. 18, 2020 RT ACC RT ACC RT ACC RT Description WLP WLP WLPWLP WLP WLP WLP WLP WLP Appearance CCS CCS CCS CCS CCS CCS CCS CCS CCSpH (100 mg/mL) 9.2-10.2 10.0 10.0 10.0 10.0 10.0 9.7 9.7 9.8 % Assay90.0%-105.0% 100.3 99.7 99.3 100.0 99.6 99.1 98.3 99.8  PhenobarbitalSodium Related NMT 0.2% BQL ND ND ND ND ND ND ND Substances 2EPMM PBGNMT 0.2% ND ND ND ND ND ND ND ND PEAU NMT 0.2% BQL BQL BQL BQL BQL BQLBQL BQL Any NMT 0.2% BQL BQL BQL BQL BQL BQL BQL BQL unspecifieddegradation product Total NMT 1.5% BQL BQL BQL BQL BQL BQL BQL BQLdegradation products Water Content NMT 3.0% 0.7 0.7 0.9 0.9 1.1 1.0 1.31.4 by Karl Fischer Note Book Reference 192039-150 192045- 23 192045-135192051-84 192099- to 158 to 45 to 151 to 122 157, 159, 167, 170 DOA:Date of Analysis; WLP: White Lyophilized Powder; CCS: Clear ColorlessSolution, 1 minute after reconstitution; ND: Not Detected; NMT: Not MoreThan; BQL: Below Quantitation Limit (0.05%) 2EPMM:2-Ethyl-2-Phenylmalonamide, monohydrate; PBG: α-Phenylbutyrylguanidine;PEAU: Phenylethylacetylurea;

TABLE 12 Product Name Phenobarbital Sodium for Injection USP, 200mg/vial Batch#NPH2031A Fill Volume: 2.1 mL Date Manufactured: Jun. 3,2020 Vial Make 10 mL Tubular Serum Bottle clear Date Initiated: Jun. 9,2020 API Mfg. Phenobarbital Sodium, USP Grade P, C-IV:, Stopper Used 20mm Igloo Bromobutyl Lyophilization Rubber stopper Flip Off Seals: 20 mmRed Plastic top flip off with Aluminum Seal, Lot#224203 ConfigurationInverted Stability 25° C., 60% RH (RT) & 40° C., 75% RH (ACC) Initial 3Months DOA: DOA: Sep. 4, 2020 Test Spec. Jun. 5, 2020 RT ACC DescriptionWLP WLP WLP WLP Appearance CCS CCS CCS CCS pH (20 mg/mL) 9.2-10.2 9.99.6 9.7 % Assay 90.0-105.0 100.1  98.1 101.0 Phenobarbital SodiumRelated NMT 0.2% ND ND ND Substances (% Area) 2EPMM PBG NMT 0.2% ND NDND PEAU NMT 0.2% BQL BQL BQL Any NMT 0.2% BQL 0.06 0.05 unspecifieddegradation product @RRT 0.37 Total NMT 1.5% BQL 0.06 0.05 degradationproducts Water Content NMT 3.0% 1.1 1.0 1.3 by Karl Fischer Note BookReference 192045-163 to 192051- 84 to 189 122 DOA: Date of Analysis;WLP: White Lyophilized Powder; CCS: Clear Colorless Solution, 1 minuteafter reconstitution; ND: Not Detected; NMT: Not More Than; BQL: BelowQuantitation Limit (0.05%) 2EPMM: 2-Ethyl-2-Phenylmalonamide,monohydrate; PBG: α-Phenylbutyrylguanidine; PEAU: Phenylethylacetylurea

TABLE 13 Product Name Phenobarbital Sodium for Injection USP, 200mg/vial (Phenobarbital Sodium and 9% Saline/vial) Batch#NPH2031B FillVolume: 2.1 mL Date Manufactured: Jun. 3, 2020 Vial Make 10 mL TubularSerum Bottle clear Date Initiated: Jun. 9, 2020 API Mfg. PhenobarbitalSodium, USP Grade P, C-IV Re test Date: Jul. 20, 2024 Stopper Used 20 mmLyophilization Rubber stopper Flip Off Seals: 20 mm Red Plastic top flipoff with Aluminum Seal Configuration Inverted Stability 25° C., 60% RH(RT) & 40° C., 75% RH (ACC) Initial 3 Months 6 Months 12 months DOA:DOA: Sep. 4, 2020 DOA: Dec. 11, 2020 DOA: 11 Apr. 2022 Test Spec. Jun.5, 2020 RT ACC RT ACC RT Description WLP WLP WLP WLP WLP WLP WLPAppearance CCS CCS CCS CCS CCS CCS CCS pH (20 mg/mL) 9.2-10.2 9.8 9.59.5 9.6  9.6  9.5 % Assay 90.0-105.0 103.3  102.8 103.6 104.0   103.7  99.9  Phenobarbital Sodium Related Substances NMT 0.2% BQL ND ND ND NDND (% Area) 2EPMM PBG NMT 0.2% ND ND ND ND ND ND PEAU NMT 0.2% BQL BQLBQL BQL 0.05 (BQL) Any unspecified NMT 0.2% BQL 0.06 0.05 0.06 0.05(BQL) degradation product @RRT 0.37 Total degradation NMT 1.5% BQL 0.060.05 0.06 0.10 BQL products Water Content NMT 4.0% 0.8 0.9 1.1 NP NP 1.2by Karl Fischer Note Book Reference 192045-163 192051- 84 192067-78192034-137 to 189 to 122 to 88 DOA: Date of Analysis; WLP: WhiteLyophilized Powder; CCS: Clear Colorless Solution, 1 minute afterreconstitution; ND: Not Detected; NMT: Not More Than; BQL: BelowQuantitation Limit (0.05%) 2EPMM: 2-Ethyl-2-Phenylmalonamide,monohydrate; PBG: α-Phenylbutyrylguanidine; PEAU: Phenylethylacetylurea

Data for Osmolality, Particulate matter and assay for Sodium Chloride isdisclosed in Table 14.

TABLE 14 Phenobarbital Sodium for Injection, USP, 200 mg/vial in Wateror Sodium Chloride - Osmolality, Particulate Matter and Assay for SodiumChloride. Assay of Sodium Osmolality* Particulate Matter Chloride(mOsmol/Kg) (By Light Obscuration method) (titrimetric; Ref(Specification: Ref: USP<788> USP monograph To be reported) ≥10 μM ≥25μM for Sodium Reconstitution Specification: Specification: ChlorideInjection in 10 mL (NMT 6000 (NMT 600 Specification: Batch No (20 mg/mL)particles/vial) particles/vial (95.0%-105.0%) NPH2031A (200 148 80.00.67 NA mg/vial) NPH2031B (200 372 171 1.33 99.99 mg/vial)(Phenobarbital Sodium 200 mg, 9% Sodium Chloride) Water -Blank 0 NA NANA 0.9% Sodium Chloride 270 NA NA NA

Lyophilization parameters used for the 65 mg/vial, 100 mg/vial, 130mg/vial and 200 mg/vial are disclosed in Table 15.

TABLE 15 Lyophilization Cycle parameters. Lyophilization Cycle 1*Lyophilization Cycle 2** Condenser Hold Condenser Hold set point VacuumTemperature Time set point Vacuum Temperature Time Section Phase C.[mTorr] ° C. [mm] ° C. [mTorr] ° C. [mm] 1 Loading off 5 n.a. off −5n.a. 2 Freezing off −5  5 off −5 10 3 Freezing off −50 60 off −52 120(Ramp) 4 Freezing off −10 30 off −52 180 (Hold) 5 Freezing off −50 120 n.a. n.a. n.a. 6 Evacuation −50 75 −50 30 −60 50 −45 30 7 Primary 75 −50400 50 −42 100 drying (Hold) (Ramp) 8 Primary 75 −45 350 50 −42 1120drying (Hold) (Hold) 9 Primary 75 −43 300 n.a. n.a. n.a. drying (Hold)10 Primary 75 −35 250 n.a. n.a. n.a. drying (Hold) 11 Primary 75 −20 60n.a. n.a. n.a. drying (Hold) 12 Primary 75 0 120 n.a. n.a. n.a. drying(Hold) 13 secondary 75 50 120 50 35 100 drying (Hold) (Ramp) 14secondary 75 25 120 50 35 420 drying (Hold) (Hold) Lyophilization Cycle1*: 65 mg/vial, 130 mg/vial and 100 mg/vial samples disclosed in Tables1, 2, 3, 16 and 17 Lyophilization Cycle 2**: NPH2031A (200 mg/vialPhenobarbital Sodium) and NPH2031B (200 mg/vial in Phenobarbital Sodiumand 90 mg/vial Sodium Chloride, NPH2020 (65 mg/vial) and NPH2021 (130mg/vial)

Six months stability data for Phenobarbital Sodium for Injection USP,100 mg/vial is included in Table 16.

TABLE 16 Product Name Phenobarbital Sodium for Injection USP, 100mg/vial Batch#NPH1980 Fill Volume: 1.0 mL Date Manufactured: Sep. 25,2019 Vial Make 5 mL Clear Untreated 5 mL Clear Treated Date Initiated:Sep. 30, 2019 API Mfg. Phenobarbital Sodium, USP Grade P, C-IV:, Re testDate: Stopper Used 13 mm Grey Bromobutyl 2 leg Lyophilization Rubberstopper Flip Off Seals: 13 mm Red Plastic top flip off with AluminumSeal Configuration Inverted Stability 25° C., 60% RH (RT) & 40° C., 75%RH (ACC) 1 Month 3 Months 6 Months Initial DOA: Oct. 31, 2019 DOA: Jan.10, 2020 DOA: Apr. 8, 2020 DOA: Sep. 27, 2019 RT ACC RT ACC RT ACC TestSpec. UT T UT T UT T UT T UT T UT T UT T Description WLP WLP WLP WLP WLPWLP WLP WLP WLP WLP WLP WLP WLP WLP WLP Appearance CCS CCS CCS CCS CCSCCS CCS CCS CCS CCS CCS CCS CCS CCS CCS pH (100 mg/mL) 9.2-10.2 9.7 9.79.6 9.7 9.6 9.6 9.7 9.7 9.7 9.7 9.8 9.9 9.8 9.9 % Assay 90.0%-105.0%98.4 97.6 98.6 99.0 99.0 100.4 99.1  99.7  97.0  96.9  97.7  98.3  97.2 97.6  Phenobarbital Sodium % Impurity NMT 0.2% ND ND ND ND ND ND BQL BQLBQL BQL ND ND ND ND 2EPMM PBG NMT 0.2% ND ND ND ND ND ND ND ND ND ND NDND ND ND PEAU NMT 0.2% BQL BQL BQL BQL BQL BQL BQL BQL BQL BQL BQL BQLBQL BQL Any unspecified NMT 0.2% 0.06 0.06 0.05 0.05 0.05 0.05 BQL BQLBQL BQL BQL BQL BQL BQL degradation product Total Impurities NMT 2.0%0.06 0.06 0.05 0.05 0.05 0.05 BQL BQL BQL BQL BQL BQL BQL BQL WaterContent NMT 3.0% 0.7 0.6 1.0 0.9 1.1 1.3 1.2 1.1 1.6 1.4 1.6 1.2 1.5 2.0by Karl Fischer Note Book Reference 192031-91 to 101 192031-169 to 183192039-44 to 52 192045-36 to 85 DOA: Date of Analysis WLP: WhiteLyophilized powder; CCS: Clear Colorless Solution, 1 minute afterreconstitution; UT: Untreated; T: Treated ND: Not Detected; NMT: NotMore Than; BQL: Below Quantitation Limit (0.05%) 2EPMM:2-Ethyl-2-Phenylmalonamide, monohydrate; PBG: α-Phenylbutyrylguanidine;PEAU: Phenylethylacetylurea

TABLE 17 Phenobarbital Sodium for Injection, USP, 100 mg/vialFormulation Details. NPH1980 (Bulk Solution at 100 mg · mL PhenobarbitalSodium) Compounding at 2-8° Fill Volume 1.0 mL mg/mL Batch (afterQuantity Ingredients reconstitution) mg/vial (100 mL) Phenobarbital 20100 10.113 g Sodium WFI/DI qs qs Qs to 100 mL or 103.3 g Water Diluentsthat can be used: Water for Injection, 0.9% Saline or 5% DextroseProposed Reconstitution Volume: 5 mL

Representative chromatograms and/or corresponding calculations are shownin FIGS. 2A-2H, 3A-3L, 4A-4L, and Tables 2 and 3.

The results for further extended stability studies are presented belowin which commercially available phenobarbital sodium, USP Grade P wasdissolved in water to produce an aqueous solution having a pH of between9.2 and 10.2, and in which the aqueous solution was then filled intolyophilization vials in an amount of between 65 mg per vial and 200 mgper vial at a maximum concentration of phenobarbital of 100 mg/ml.Lyophilization was performed as in Table 15 noted above. Stabilitystudies were then performed at standard conditions (RT: 25° C., 60%relative humidity) and accelerated conditions (ACC: 40° C., 75% relativehumidity) for a period of up to 37 months.

After the storage conditions indicated in the tables below, thelyophilized product was then dissolved in water (or saline or dextrose)and subjected to analysis for degradation products of phenobarbital.Specifically, the inventors investigated the levels of degradation byquantifying the remaining phenobarbital and by quantifying2-ethyl-2-phenylmalonamide (2EPMM), alpha-phenylbutyrylguanidine (PBG),and phenylethylacetylurea (PEAU), as well as other unspecifieddegradation products. Remarkably, and as can be seen from the databelow, the lyophilized amorphous phenobarbital had a storage stabilityat 25° C. and 60% relative humidity for up to and beyond three years anddid readily dissolve upon reconstitution.

Table 18 depicts exemplary results for 65 mg per vial phenobarbitalsodium stored at standard and accelerated conditions where under alltested conditions 2EPMM, PBG, and PEAU remained undetectable or belowquantitation limit (0.05%). Likewise, unspecified degradation productsand total degradation products remained below quantitation limit(0.05%).

TABLE 18 (65 mg/vial, up to 24 months) Product Name Phenobarbital Sodiumfor Injection USP, 65 mg/vial Batch#NPH2020 Fill Volume: 1.05 mL DateManufactured: Jan. 20, 2020 Vial Make 2 mL Fiolax Clear Blow Back VialDate Initiated: Feb. 14, 2020 API Mfg. Phenobarbital Sodium, USP GradeP, C-IV: Stopper Used 13 mm Grey Bromobutyl 2 leg Lyophilization Rubberstopper

 Flip Off Seals: 13 mm Red Plastic top flip off with Aluminum SealConfiguration Inverted Stability 25° C., 60% RH (RT) & 40° C., 75% RH(ACC) 24 Month Initial 1 Month 3 Months 6 Months DOA: DOA: DOA: Mar. 2

, 2020 DOA: May 20, 2020 DOA: Sep. 4, 2020 18 Mar. 2022 Test Spec. Feb.18, 2020 RT ACC RT ACC RT ACC RT Description WLP WLP WLP WLP WLP WLP WLPWLP WLP Appearance CCS CCS CCS CCS CCS CCS CCS CCS CCS pH (65 mg/mL)9.2-10.2 9.9 9.9 9.9 9.9 9.9 9.6 9.

9.7 % Assay 90.0%-105.0% 98.6  101.6  101.5  101.3  100.3  100.0  101.2101.2  Phenobarbital Sodium Related Substances NMT 0.2% BQL ND ND ND NDND ND ND 2EPMM PBG NMT 0.2% ND ND ND ND ND ND ND ND PEAU NMT 0.2% BQLBQL BQL BQL BQL BQL BQL BQL Any unspecified NMT 0.2% BQL BQL BQL BQL BQLBQL BQL BQL degradation product Total degradation products NMT 1.5% BQLBQL BQL BQL BQL BQL BQL BQL Water Content by Karl NMT 3.0% 0.7 0.9 1.31.4 1.5 1.6  2.1 1.8 Fischer Note Book Reference 192039- 192045-23192045-135 192051-

4

150 to 158 to

to 151 to 122 DOA: Date of Analysis; WLP: White Lyophilized Powder; CCS:Clear Colorless Solution, 1 minute after reconstitution; ND: NotDetected; NMT: Not More Than; BQL: Below Quantitation Limit (0.05%)2EPMM: 2-Ethyl-2-Phenylmalonamide, monohydrate; PBG:α-Phenylbutyrylguanidine; PEAU: Phenylethylacetylurea;

indicates data missing or illegible when filed

Table 19 depicts exemplary results for 65 mg per vial phenobarbitalsodium stored at standard and accelerated conditions where under alltested conditions 2EPMM, PBG, and PEAU remained undetectable or belowquantitation limit (0.05%). Likewise, unspecified degradation productsand total degradation products remained below quantitation limit (0.05%)or barely above quantitation limit.

TABLE 19 (65 mg/vial, up to 37 months) Product Name Phenobarbital Sodiumfor Injection USP, 65 mg/vial Batch#NPH1907 Date Manufactured: Feb. 21,2019 Vial Make 2 mL Fiolax Clear Blow Back Vial Date Initiated: Feb. 25,2019 API Mfg. Phenobarbital Sodium, USP Grade P, C-IV: Stopper Used 13mm Grey Bromobutyl 2 leg Lyophilization Rubber stopper Flip Off Seals:13 mm Red Plastic top flip off with Aluminum Seal Configuration InvertedStability 25° C. ± 2° C., 60% RH ± 5% (RT) & 40° C., ±

° C., 75% RH ± 5% (ACC) 15 Months Initial 1 Month 3 Months 6 Months DOA:

DOA: DOA: Mar. 28, 2019 DOA: May 24, 2019 DOA:

. 2

, 2019

. 12, 2020 Months Test Spec. Feb. 25, 2019 RT ACC RT ACC RT ACC *RT **RT

CCS CCS CCS CCS CCS CCS CCS CCS CCS

ND ND ND ND ND ND ND ND

ND ND ND ND ND ND ND ND

ND BQL

BQL

BQL

BQL BQL

BQL

1.1

1.6 1.

1.

indicates data missing or illegible when filed

Table 20 depicts exemplary results for 100 mg per vial phenobarbitalsodium stored at standard and accelerated conditions where under alltested conditions 2EPMM, PBG, and PEAU remained undetectable or belowquantitation limit (0.05%). Likewise, unspecified degradation productsand total degradation products remained below quantitation limit (0.05%)or barely above quantitation limit.

TABLE 20 (100 mg/vial, up to 30 months) Product Name PhenobarbitalSodium for Injection USP, 100 mg/vial Batch#NPH19

0 Fill Volume: 1.0 mL Date Manufactured: Sep. 2

, 2019 Vial Make

 mL Clear Untreated 5 mL Clear Treated Date Initiated: Sep. 30, 2019 APIMfg. Phenobarbital Sodium, USP Grade P, C-IV: Stopper Used 13 mm GreyBromobutyl 2 leg Lyophilization Rubber stopper Flip Off Seals: 13 mm RedPlastic top flip off with Aluminum Seal Lot# NA Configuration InvertedStability 25° C., 60% RH (RT) & 40° C., 75% RH (ACC) 1 Month 3 MonthsInitial DOA: Oct. 31, 2019 DOA: Jan. 10, 2020 DOA: Sep. 27, 2019 RT ACCRT ACC T

Spec. UT T UT T UT T UT T UT T Description WLP WLP WLP WLP WLP WLP WLPWLP WLP WLP WLP Appearance CCS CCS CCS CCS CCS CCS CCS CCS CCS CCS CCSpH (100 mg/mL) 9.2-10.2 9.7 9.7  9.6 9.7 9.6  9.6 9.7 9.7 9.7 9.7 %Assay of Phenobarbital 90.0%-105.0% 98.4 97.

9

.

99.0  9

.0 100.4  99.1  98.7 

.0

.9 % Impurity NMT 0.2% ND ND ND ND ND ND BQL BQL BQL BQL 2EPMM PBG NMT0.2% ND ND ND ND ND ND ND ND ND ND PEAU NMT 0.2% BQL BQL ND ND BQL BQLBQL BQL BQL BQL Any unspecified NMT 0.2% 0.0

0.0

0.0

0.0

0.05 0.0

BQL BQL BQL BQL degradation product @RRT0.37 Total Impurities NMT 1.5%0.0

0.08  0.05  0.05 0.05  0.05 BQL BQL BQL BQL Water Content by Karl NMT4.0% 0.7 0.

1.0 0.

1.1  1.1 1.2 1.1 1.

1.4 Fischer Note Book Reference 192031-91 192031-1

192039-44 to 101 to 183 to 52

 Months 30 Months DOA: Apr.

, 2020 DOA: 18 Mar. 2022 RT ACC RT T

Spec. UT T UT T UT T Description WLP WLP WLP WLP WLP WLP WLP AppearanceCCS CCS CCS CCS CCS CCS CCS pH (100 mg/mL) 9.2-10.2 9.8

.9 9.8 9.9 9.

9.

% Assay of Phenobarbital 90.0%-105.0% 97.7  98.3  97.2  97.6  99.2 100.0 % Impurity NMT 0.2% BQL BQL BQL BQL BQL BQL 2EPMM PBG NMT 0.2% NDND ND ND ND ND PEAU NMT 0.2% BQL BQL BQL BQL BQL BQL Any unspecified NMT0.2% BQL BQL BQL BQL BQL BQL degradation product @RRT0.37 TotalImpurities NMT 1.5% BQL BQL BQL BQL BQL BQL Water Content by Karl NMT4.0% 1.6 1.2 1.5 2.

2.1  1.8 Fischer Note Book Reference 192045-4

192039- to 85 157, 15

, 1

, 170157, 1

9, 1

7, 1

DOA: Date of Analysis; WLP: White Lyophilized Powder; CCS: ClearColorless Solution, 1 minute after reconstitution; UT: Untreated; T:Treated ND: Not Detected; NMT: Not More Than; BQL: Below QuantitationLimit (0.05%) 2EPMM: 2-Ethyl-2-Phenylmalonamide, monohydrate; PBG:α-Phenylbutyrylguanidine; PEAU: Phenylethylacetylurea

indicates data missing or illegible when filed

Table 21 depicts exemplary results for 130 mg per vial phenobarbitalsodium stored at standard and accelerated conditions where under alltested conditions 2EPMM, PBG, and PEAU remained undetectable or belowquantitation limit (0.05%). Likewise, unspecified degradation productsand total degradation products remained at or below quantitation limit(0.05%).

TABLE 21 (130 mg/vial, up to 37 months) Product Name PhenobarbitalSodium for Injection USP, 130 mg/vial Batch#NPH1912 Date Manufactured:Feb. 23, 2019 Vial Make 2 mL Fiolax Clear Blow Back Vial Date Initiated:Feb. 25, 2019 API Mfg. Phenobarbital Sodium, USP Grade P, C-IV: StopperUsed 13 mm Grey Bromobutyl 2 leg Lyophilization Rubber stopper Flip OffSeals: 13 mm Red Plastic top flip off with Aluminum Seal ConfigurationInverted Stability 25° C. ± 2° C., 60% RH ± 5% (RT) & 40° C., ± 2° C.,75% RH ± 5% (ACC) 1 Month 3 Months Initial DOA: Mar. 28, 2019 DOA: May24, 2019 Test Spec. DOA: Feb. 28, 2019 RT ACC RT ACC Description WLP WLPWLP WLP WLP WLP Appearance CCS CCS CCS CCS CCS CCS pH (10

 mg/mL) 9.2-10.2 9.8 10.0 9.9 9.

9.

% Assay 90.0%-105.0% 98.3  97.1 98.3  100.1  97.9  Phenobarbital SodiumRelated Substances NMT 0.2% ND ND ND ND ND 2EPMM PBG NMT 0.2% ND ND NDND ND PEAU NMT 0.2% ND BQL BQL BQL BQL Any unspecified NMT 0.2% BQL BQLBQL BQL BQL degradation product Total Degradation products NMT 1.5% BQLBQL BQL BQL BQL Water Content by Karl NMT 4.0% 0.

 1.1 1.2 1.1 1.3 Fischer Note Book Reference 192019- 192019- 192025- 41,44, 45, 47 122, 12

, 125, 12

24, 30, 31, 34 6 Months 15 Months 37 Months DOA: Aug. 28, 2019 DOA: Jun.12, 2020 DOA: 18 Mar. 2022 Test Spec. RT ACC RT RT Description WLP WLPWLP WLP WLP Appearance CCS CCS CCS CCS CCS pH (10

 mg/mL) 9.2-10.2 9.8 9.8 10.0 9.

% Assay 90.0%-105.0% 98.

99.4 98.3 99.2 Phenobarbital Sodium Related Substances NMT 0.2% ND ND NDND 2EPMM PBG NMT 0.2% ND ND ND ND PEAU NMT 0.2% BQL BQL BQL BQL Anyunspecified NMT 0.2% BQL 0.05 BQL BQL degradation product TotalDegradation products NMT 1.5% BQL 0.05 BQL BQL Water Content by Karl NMT4.0% 1.

1.4 1.4  1.5 Fischer Note Book Reference 192031- 192051-2 192099- 8, 12,14, 16, 17, 1

to 11 157, 159, 1

7, 1

DOA: Date of analysis; WLP: White Lyophilized Powder; CCS: ClearColorless Solution, 1 minute after reconstitution; ND: Not Detected;NMT: Not More Than; BQL: Below Quantitation Limit (0.05%) 2EPMM:2-Ethyl-2-Phenylmalonamide, monohydrate; PBG: α-Phenylbutyrylguanidine;PEAU: Phenylethylacetylurea

indicates data missing or illegible when filed

Table 22 depicts exemplary results for 130 mg per vial phenobarbitalsodium stored at standard and accelerated conditions where under alltested conditions 2EPMM, PBG, and PEAU remained undetectable or belowquantitation limit (0.05%). Likewise, unspecified degradation productsand total degradation products remained below quantitation limit(0.05%).

TABLE 22 (130 mg/vial, up to 24 months) Product Name PhenobarbitalSodium for Injection USP, 130 mg/vial Batch#NPH2021 Fill Volume: 1.05 mLDate Manufactured: Jan. 20, 2020 Vial Make 2 mL Fiolax Clear Blow BackVial Date Initiated: Feb. 14, 2020 API Mfg. Phenobarbital Sodium, USPGrade P, C-IV: Stopper Used 13 mm Grey Bromobutyl 2 leg LyophilizationRubber stopper Flip Off Seals: 13 mm Red Plastic top flip off withAluminum Seal Configuration Inverted Stability 25° C., 60% RH (RT) & 40°C., 75% RH (ACC) 1 Month 3 Months 6 Months 24 Months Initial DOA: DOA:DOA: DOA: BOA: Mar. 23, 2020 May 20, 2020 Sep. 4, 2020 18 Mar. 2022 TestSpec. Feb. 18, 2020 RT ACC RT ACC RT ACC RT Description WLP WLP WLP WLPWLP WLP WLP WLP WLP Appearance CCS CCS CCS CCS CCS CCS CCS CCS CCS pH(100 mg/mL) 9.2-10.2 10.0  10.0  10.0 10.0  10.0 9.7 9.7 9.8 % Assay90.0%-105.0% 100.3  99.

99.3 100.0  99.6 99.1  98.3  99.8  Phenobarbital Sodium RelatedSubstances NMT 0.2% BQL ND ND ND ND ND ND ND 2EPMM PBG NMT 0.2% ND ND NDND ND ND ND ND PEAU NMT 0.2% BQL BQL BQL BQL BQL BQL BQL BQL Anyunspecified NMT 0.2% BQL BQL BQL BQL BQL BQL BQL BQL degradation productTotal degradation products NMT 1.5% BQL BQL BQL BQL BQL BQL BQL BQLWater Content by Karl NMT 4.0% 0.7 0.7  0.9 0.9  1.1 1.0 1.3 1.4 FischerNote Book Reference 192039-150 192045- 23 192045-135 192051-84 192099-to 158 to 45 to 151 to 122 157, 159, 167, 170 DOA: Date of Analysis;WLP: White Lyophilized Powder; CCS: Clear Colorless Solution, 1 minuteafter reconstitution; ND: Not Detected; NMT: Not More Than; BQL: BelowQuantitation Limit (0.05%) 2EPMM: 2-Ethyl-2-Phenylmalonamide,monohydrate; PBG: α-Phenylbutyrylguanidine; PEAU: Phenylethylacetylurea;

indicates data missing or illegible when filed

Table 22 depicts exemplary results for 200 mg per vial phenobarbitalsodium stored at standard and accelerated conditions where under alltested conditions 2EPMM, PBG, and PEAU remained undetectable or belowquantitation limit (0.05%). Likewise, unspecified degradation productsand total degradation products remained near or below quantitation limit(0.05%).

TABLE 22 (200 mg/vial, up to 22 months) Product Name PhenobarbitalSodium for Injection USP, 200 mg/vial Batch#NPH2031A Fill Volume: 2.1 mLDate Manufactured: Jun. 3, 2020 Vial Make 10 mL Tubular Serum Bottleclear Date Initiated: Jun. 9, 2020 API Mfg. Phenobarbital Sodium, USPGrade P, C-IV: Stopper Used 20 mm Igloo Bromobutyl Lyophilization Rubberstopper Flip Off Seals: 20 mm Red Plastic top flip off with AluminumSeal Configuration Inverted Stability 25° C., 60% RH (RT) & 40° C., 75%RH (ACC) 3 Months 6 months 11 mouths Initial DOA: Sep. 4, 2020 DOA: Dec.11, 2020 DOA: 11 Apr. 2022 Test Spec. DOA: Jun. 5, 2020 RT ACC RT ACC RTDescription WLP WLP WLP WLP WLP WLP WLP Appearance CCS CCS CCS CCS CCSCCS CCS pH (20 mg/mL) 9.2-10.2 9.9 9.6 9.7 9.7 9.7 9.5 % Assay90.0-105.0 100.1  98.1 101.0 100.8 102.5 98.2  Phenobarbital SodiumRelated Substances (% NMT 0.2% ND ND ND ND ND ND Area) 2EPMM PBG NMT0.2% ND ND ND ND ND ND PEAU NMT 0.2% BQL BQL BQL BQL BQL BQL Anyunspecified NMT 0.2% BQL 0.06 0.05 0.06 0.05 (BQL) degradation product@RRT 0.37 Total degradation products NMT 1.5% BQL 0.06 0.05 0.06 0.05(BQL) Water Content by Karl NMT 3.0% 1.1 1.0 1.3 1.2 1.6 1.5 FischerNote Book Reference 192045-163 to 189 192051- 84 to 122 192067-78 to 88192034-137 DOA: Date of Analysis; WLP: White Lyophilized Powder; CCS:Clear Colorless Solution, 1 minute after reconstitution; ND: NotDetected; NMT: Not More Than; BQL: Below Quantitation Limit (0.05%)2EPMM: 2-Ethyl-2-Phenylmalonamide, monohydrate; PBG:α-Phenylbutyrylguanidine; PEAU: Phenylethylacetylurea

Table 23 depicts exemplary results for 200 mg per vial phenobarbitalsodium stored at standard and accelerated conditions where under alltested conditions 2EPMM, PBG, and PEAU remained undetectable or belowquantitation limit (0.05%). Likewise, unspecified degradation productsand total degradation products remained near or below quantitation limit(0.05%). In contrast to Table 22, this formulation was filled into thevial using a 9% saline solution. The final concentration of the salineafter reconstitution in 10 mL of water for injection will be 0.9%.

TABLE 23 (200 mg/vial, up to 12 months) Product Name PhenobarbitalSodium for Injection USP, 200 mg/vial (Phenobarbital Sodium and 9%Saline/vial) Batch#NPH2031B Fill Volume: 2.1 mL Date Manufactured: Jun.3, 2020 Vial Make 10 mL Tubular Serum Bottle clear Date Initiated: Jun.9, 2020 API Mfg. Phenobarbital Solution, USP Grade P, C-IV: Stopper Used20 mm Igloo Bromobutyl Lyophilization Rubber stopper Flip Off Seals: 20mm Red Plastic top flip off with Aluminum Seal Configuration InvertedStability 25° C., 60% RH (RT) & 40° C., 75% RH (ACC) 3 Months 6 Months12 months Initial DOA: Sep. 4, 2020 DOA: Dec. 11, 2020 DOA: 11 Apr. 2022Test Spec. DOA: Jun. 5, 2020 RT ACC RT ACC RT Description WLP WLP WLPWLP WLP WLP WLP Appearance CCS CCS CCS CCS CCS CCS CCS pH (20 mg/mL)9.2-10.2 9.8 9.5 9.5 9.6  9.6  9.5 % Assay 90.0-105.0 103.3  102.8 103.6104.0   103.7   99.9  Phenobarbital Sodium Related Substances NMT 0.2%BQL ND ND ND ND ND (% Area) 2EPMM PBG NMT 0.2% ND ND ND ND ND ND PEAUNMT 0.2% BQL BQL BQL BQL 0.05 (BQL) Any unspecified NMT 0.2% BQL 0.060.05 0.06 0.05 (BQL) degradation product @RRT 0.37 Total degradationproducts NMT 1.5% BQL 0.06 0.05 0.06 0.10 BQL Water Content by Karl NMT4.0% 0.8 0.9 1.1 NP NP 1.2 Fischer Note Book Reference 192045-163192051- 84 192867-78 192034-137 to 189 to 122 to 88 DOA: Date ofAnalysis; WLP: White Lyophilized Powder; CCS: Clear Colorless Solution,1 minute after reconstitution; ND: Not Detected; NMT: Not More Than;BQL: Below Quantitation Limit (0.05%) 2EPMM: 2-Ethyl-2-Phenylmalonamide,monohydrate; PBG: α-Phenylbutyrylguanidine; PEAU: Phenylethylacetylurea

To further investigate if the nature of the reconstitution solutionwould have an adverse effect on stability after reconstitution, theinventors used 65 mg of lyophilized amorphous phenobarbital in a vialand dissolved the lyophilized material with 0.9% or 5% dextrose to afinal concentration of 1 mg/ml. In an additional set of experiments, theinventors used 130 mg of lyophilized amorphous phenobarbital in a vialand dissolved the lyophilized material with 0.9% or 5% dextrose or waterto a final concentration of 20 mg/ml. The so reconstituted solutionswere then kept under refrigeration (2-8° C.) or at standard conditions(25° C.) for the indicated times in the tables below. The levels ofdegradation were determined by quantifying the remaining phenobarbitaland by quantifying 2-ethyl-2-phenylmalonamide (2EPMM),alpha-phenylbutyrylguanidine (PBG), and phenylethylacetylurea (PEAU), aswell as other unspecified degradation products. Once more, under alltested conditions, the stability of the so reconstituted compositionswas unexpectedly high.

Table 24 depicts exemplary results for 65 mg per vial phenobarbitalsodium reconstituted with 5% dextrose to 1 mg/ml that was stored underrefrigeration or at standard conditions. Under all tested conditions2EPMM, PBG, and PEAU remained undetectable or below quantitation limit(0.05%). Likewise, unspecified degradation products and totaldegradation products remained near or below quantitation limit (0.05%).

TABLE 24 (65 mg/vial, reconstituted to 1 mg/ml in 5% dextrose, up to 24hrs) Admixture Study of Phenobarbital Sodium for Injection USP, 65mg/vial (NPB2020) (1 mg/mL dilution in 5% Dextrose) Time 1 hr 2.5 hr 4hrs 6 hrs 9 hrs 24 hrs 2-8 2-8 2-8 2-8 2-8 2-8 Specifications TestsInitial C. RT C. RT C. RT C. RT C. RT C. RT Complies Appearance CCS CCSCCS CCS CCS CCS CCS CCS CCS CCS CCS CCS CCS pH pH (1 mg/mL 8.8 8.8 8.88.8 8.8 8.8 8.7 8.8 8.7  8.7 8.7  8.7 8.5 To be reported dilution) AssayAssay of 95.4 95.7  95.6  95.9  95.4  95.9  97.1  95.6  95.9  94.6 97.1  95.1 96.1 90%-105% Phenobarbital Sodium 2EPMM Related SubstancesND ND ND ND ND ND BQL ND ND ND BQL ND BQL NMT0.2% (% Area) 2EPMM PEG PBGND ND ND ND ND ND ND ND ND ND ND ND ND NMT0.2% PEAU PEAU ND ND BQL BQLBQL ND BQL BQL ND BQL BQL BQL BQL NMT0.2% Unspecified Any unspecified NDND BDL ND BDL ND BDL ND 0.02 BDL 0.02 BDL 0.03 degradation degradationproduct product @ RRT 0.29 NMT 0.2% Any unspecified 0.01  0.01  0.01 0.01  0.01  0.01  0.02  0.01 0.02  0.01 0.03 0.02 0.05 degradationproduct @ RRT 0.37 Osmolality Osmolality 254 NP Np NP NP NP NP NP NP NPNP 255 253 To be reported (mOsmol/Kg) NMT 1.5% Total degradation BQL BQLBQL BQL BQL BQL BQL BQL BQL BQL BQL BQL 0.08 products CCS: Clearcolorless solution; ND: Not detected; BDL: Below detection limit; BQL:Below Quantitation limit; NP: Not performed; 2EPMM:2-ethylphenylmalonamide monohydrate; PBG: Phenylbutyrylguanidine; PEAU:Phenylethylacetylurea

Table 25 depicts exemplary results for 65 mg per vial phenobarbitalsodium reconstituted with 0.9% saline to 1 mg/ml that was stored underrefrigeration or at standard conditions. Under all tested conditions2EPMM, PBG, and PEAU remained undetectable or below quantitation limit(0.05%). Likewise, unspecified degradation products and totaldegradation products remained near or below quantitation limit (0.05%).

TABLE 25 (65 mg/vial, reconstituted to 1 mg/ml in 0.9% saline, up to 24hrs) Admixture study of Phenobarbital Sodium for Injection USP, 65mg/vial (NPH2020) (1 mg/mL) dilution in 0.9% Saline) Time 1 hr 2.5 hrs 4hrs 6 hrs 9 hrs 24 hrs 2-8 2-8 2-8 2-8 2-8 2-8 Specifications TestInitial C. RT C. RT C. RT C. RT C. RT C. RT Complies Description CCS CCSCCS CCS CCS CCS CCS CCS CCS CCS CCS CCS CCS pH pH (1 mg/mL 9.0 8.8 8.99.0 9.0 8.9 8.9 8.9 8.9 8.7 8.8  8.5 8.4 To be reported dilution) AssayAssay of 95.8 96   94.3  95.4  93.6  95.5  95.2  95.8  93.9  94.6  96.4 95.2 95.3 90%-105% Phenobarbital Sodium 2EPMM Related Substances ND NDND ND ND ND BQL ND ND ND BQL ND BQL NMT 0.2% (% Area) 2EPMM PBG PBG NDND ND ND ND ND ND ND ND ND ND ND ND NMT 0.2% PEAU PEAU BQL ND BQL BQLBQL BQL BQL BQL ND BQL BQL BQL BQL NMT 0.2% Unspecified degradation Anyunspecified BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL productdegradation product NMT 0.2% @ RRT 0.29 Any unspecified 0.02  0.02  0.03 0.02  0.03  0.02  0.04  0.02  0.04  0.03 0.05 0.03 0.1 degradationproduct @ RRT 0.037 NMT Total degradation BQL BQL BQL BQL BQL BQL BQLBQL BQL BQL 0.05 BQL 0.1 1.5% products Osmolality Osmolality 288 NP NPNP NP NP NP NP NP NP NP 286 287 To be reported (mOsmol/Kg) CCS: Clearcolorless solution; ND: Not detected; BQL: Below Quantitation limit; NP:Not performed; 2EPMM: 2-ethylphenylmalonamide monohydrate; PBG:Phenylbutyrylguanidine; PEAU: Phenylethylacetylurea BQL: BelowQuantitation limit (0.05%)

Table 26 depicts exemplary results for 130 mg per vial phenobarbitalsodium reconstituted with 0.9% saline to 20 mg/ml that was stored underrefrigeration or at standard conditions. Under all tested conditions2EPMM, PBG, and PEAU remained undetectable or below quantitation limit(0.05%). Likewise, unspecified degradation products and totaldegradation products remained near or below quantitation limit (0.05%).

TABLE 26 (130 mg/vial, reconstituted to 20 mg/ml in 0.9% saline, up to24 hrs) Admixture study of Phenobarbital Sodium for Injection USP, 130mg/vial (NPH2030) (20 mg/mL dilution In 0.9% Saline) Time 30 min 90 min240 min Tests Initial 2-8 C. RT 2-8 C. RT 2-8 C. RT Appearance CCS CCSCCS CCS CCS CCS CCS pH (20 mg/mL dilution) 9.4 9.4  9.3  9.4  9.3  9.4 9.4  Assay of Phenobarbital 104.1 103.6   102.4   103.3   102.7  103.2   102.9   Sodium Related Substances ND ND ND ND ND ND BQL (% Area)2EPMM PBG ND ND ND ND ND ND ND PEAU BQL BQL BQL BQL BQL BQL BQL Anyunspecified BQL 0.03 0.04 0.03 0.05 0.03 0.07 degradation product @ RRT0.37 Total degradation BQL 0.03 0.04 0.03 0.05 0.03 0.07 productsOsmolality (mOsmol/Kg) 418 NP NP NP NP NP NP Time 6 hrs 9.5 hrs 24 hrsTests 2-8 C. RT 2-8 C. RT 2-8 C. RT Appearance CCS CCS CCS CCS CCS CCSpH (20 mg/mL dilution) 9.4  9.4  9.4 9.2 9.4 9.3 Assay of Phenobarbital103.4   102.9   99.5 103.3 103.3 101.3 Sodium Related Substances ND BQLND BQL ND BQL (% Area) 2EPMM PBG ND ND ND ND ND ND PEAU BQL BQL BQL BQLBQL BQL Any unspecified 0.03 0.09 0.03 0.13 0.04 0.24 degradationproduct @ RRT 0.37 Total degradation 0.03 0.09 0.03 0.13 0.04 0.24products Osmolality (mOsmol/Kg) NP NP 420 418 417 419 CCS: Clearcolorless solution; ND: Not detected; BQL: Below Quantitation limit; NP:Not performed; 2EPMM: 2-ethylphenylmalonamide monohydrate; PBG:Phenylbutyrylguanidine; PEAU: Phenylethylacetylurea

Table 27 depicts exemplary results for 130 mg per vial phenobarbitalsodium reconstituted with 5% dextrose to 20 mg/ml that was stored underrefrigeration or at standard conditions. Under all tested conditions2EPMM, PBG, and PEAU remained undetectable or below quantitation limit(0.05%). Likewise, unspecified degradation products and totaldegradation products remained near or below quantitation limit (0.05%).

TABLE 27 (130 mg/vial, reconstituted to 20 mg/ml in 5% saline, up to 24hrs) Admixture study of Phenobarbital Sodium for Injection USP, 130mg/vial (NPH2030) (20 mg/mL dilution In 5% Dextrose) Time 30 min 90 min4 hrs Test Initial 2-8 C. RT 2-8 C. RT 2-8 C. RT Appearance CCS CCS CCSCCS CCS CCS CCS pH (20 mg/mL dilution) 9.4 9.4 9.3 9.4  9.4 9.4  9.3 Assay of Phenobarbital 101 102 100 101.5   102 100.9   101.1   SodiumRelated Substances (% Area) ND ND ND ND ND ND BQL 2EPMM PBG ND ND ND NDND ND ND PEAU BQL BQL BQL BQL BQL BQL BQL Any unspecified 0.02 0.02 0.030.03 0.04 0.03 0.06 degradation product @ RRT 0.37 Total degradationproducts 0.02 0.02 0.03 0.03 0.04 0.03 0.06 Osmolality (mOsmol/Kg) 379NP NP NP NP NP NP Time 6 hrs 9.5 hrs 14 hrs Test 2-8 C. RT 2-8 C. RT 2-8C. RT Appearance CCS CCS CCS CCS CCS CCS pH (20 mg/mL dilution) 9.3 9.3  9.4 9.3 9.4 9.4 Assay of Phenobarbital 101.7   101.6   101.6 102.1102.4 102.2 Sodium Related Substances (% Area) ND ND ND BQL ND BQL 2EPMMPBG ND ND ND ND ND ND PEAU BQL BQL BQL BQL BQL BQL Any unspecified 0.030.08 0.03 0.11 0.04 0.19 degradation product @ RRT 0.37 Totaldegradation products 0.03 0.08 0.03 0.11 0.04 0.19 Osmolality(mOsmol/Kg) NP NP 379 380 377 379 CCS: Clear colorless solution; ND: Notdetected; BQL: Below Quantitation limit; NP: Not performed; 2EPMM:2-ethylphenylmalonamide monohydrate; PBG: Phenylbutyrylguanidine; PEAU:Phenylethylacetylurea

Table 28 depicts exemplary results for 130 mg per vial phenobarbitalsodium reconstituted with water to 20 mg/ml that was stored underrefrigeration or at standard conditions. Under all tested conditions2EPMM, PBG, and PEAU remained undetectable or below quantitation limit(0.05%). Likewise, unspecified degradation products and totaldegradation products remained near or below quantitation limit (0.05%).

TABLE 28 (130 mg/vial, reconstituted to 20 mg/ml in water, up to 24 hrs)Admixture study of Phenobarbital Sodium for Injection USP, 130 mg/vial(NPH2030) (20 mg/mL dilution In Water) Time 2 hrs 4 hrs 24 hrs TestsInitial 2-8 C. RT 2-8 C. RT 2-8 C. RT Appearance CCS CCS CCS CCS CCS CCSCCS pH (20 mg/mL dilution) 9.5 9.5  9.6  9.6  9.5  9.6 9.5 Assay ofPhenobarbital Sodium 99.8 102.1   100.8   101.7   101.2   100.4 100.3Related Substances (% Area) ND ND ND ND ND ND BQL 2EPMM PBG ND ND ND NDND ND ND PEAU BQL BQL BQL BQL BQL BQL BQL Any unspecified degradation0.02 0.02 0.04 0.02 0.06 0.04 0.18 product @ RRT 0.37 Total degradationproducts 0.02 0.02 0.04 0.02 0.06 0.04 0.18 Osmolality mOsmol/Kg 158 NPNP NP NP 159 158 CCS: Clear colourless solution; ND: Not detected; BQL:Below Quantitation limit; NP: Not performed; 2EPMM:2-ethylphenylmalonamide monohydrate; PBG: Phenylbutyrylguanidine; PEAU:Phenylethylacetylurea

In further experiments, the inventors investigated if stable lyophilizedphenobarbital preparations could be prepared (in crystalline and/oramorphous forms, depending on solvent) using one or more water miscibleco-solvents. To that end, the inventors tested isopropanol,tert-butanol, ethanol, and acetonitrile as co-solvents. Most typically,the co-solvent(s) will be present in an amount of at least 1.0%, or atleast 1.5%, or at least 2.0%, or at least 2.5%, or at least 3.0%, or atleast 3.5%, or at least 4.0%, or at least 4.5%, or at least 5.0%, or atleast 5.5%, or at least 6.0%, or at least 7.0%, or at least 8.0%, or atleast 9.0%, or at least 10%, or at least 25%, or at least 50%, or atleast 75%. Therefore, suitable quantities of co-solvent(s) include thosebetween 1.0 and 3.0%, or between 2.5% and 5.0%, or between 3.0% and7.5%, or between 5.0% and 10%, or between 10% and 25%, or between 25%and 50%, or between 50% and 75%, and even higher. For example, and asshown in more detail below, the concentration of the organic co-solventwas as high as 90%. Lyophilization conditions and results are shownbelow. Tables 29A and 29B depict formulation details used in thelyophilization and stability experiments. Two formulations were preparedfor each strength (65 mg and 130 mg), one with 5% Isopropanol and theother with 5% Tertiary Butanol, and other co-solvents (here acetonitrileand ethanol) were also evaluated. The organic solvent was added afterthe Phenobarbital Sodium was dissolved in water, mixed well and adjustedto final volume with water. Later the bulk solution is filled in vialsand lyophilized using the Lyo cycle parameters shown in Table 30. Afterthe Lyo cycle was completed, the finished product was analyzed forassay, related substances, pH, moisture content and residual solvents.

TABLE 29A Formulations with t-butanol and isopropanol as organicco-solvent Bulk CAS Total volume Ingredients Solution NumberConcentration in mL (50 mL) Phenobarbital  65 mg/mL NA 65.73 mg (~65 mg3.287 g Sodium, USP C-IV Phenobarbital Sodium after LOD correction) 130mg/mL 131.469 mg (~130 mg 6.573 g Phenobarbital sodium after LODcorrection) Tertiary Butanol 5% 75-65-0 (0.05 ml or 0.039 g) 2.5 ml or1.95 g Isopropanol 67-63-0 (0.05 ml or 0.0393 g) 2.5 ml or 1.963 g DIWater/Water for N/A Q.S. to 1 mL Q.S. to 50 mL Injection

TABLE 29B Formulations with ethanol and acetonitrile as organicco-solvent Bulk Solution Formulation Total Volume prepared (100 mL BatchSize) Ingredients at 25° C. NPH22035 NPH22034 Phenobarbital 130 mg/mL13.38 g 13.38 g Sodium Ethanol Qs — Qs to total volume 100 mLAcetonitrile Qs Qs to total — volume 100 mL Water for 10% 10 ml 10 mlInjection/ DI water

TABLE 30 Lyophilization protocol Hold Time Temperature Vacuum SectionPhase [mm] ° C. [mTorr] 1 Loading n.a. 5 off 2 Freezing 5 −5 off 3Freezing 60 −50 off 4 Freezing 30 −10 off 5 Freezing 120 −50 off 6Evacuation 30 −50 75 7 Primary drying 400 −50 75 8 Primary drying 350−45 75 9 Primary drying 300 −43 75 10 Primary drying 250 −35 75 11Primary drying 60 −20 75 12 Primary drying 120 0 75 13 Secondary drying120 50 75 14 Secondary drying 120 25 75

Table 31 depicts results for 65 mg and 130 mg per vial phenobarbitalsodium (from a bulk solution containing 5% tert-butanol) stored atstandard and accelerated conditions where under all tested conditions2EPMM, PBG, and PEAU remained undetectable or below quantitation limit(0.05%). Likewise, unspecified degradation products and totaldegradation products remained near or below quantitation limit (0.05%).

TABLE 31 (65/130 mg/vial, 5% tert-butanol, up to 3 months) Product NamePhenobarbital Sodium for Injection, USP Lyophilized Powder. Bulksolution prepared in 5% Tertiary Butanol Date Manufactured: Feb. 21,2019 Date Initiated: Sep. 5, 2019 Configuration Inverted Stability 25°C., 60% RH (RT) & 40° C., 75% RH (ACC) NPH1965 (65 mg/vial) NPH1967 (130mg/vial) 3 Months 3 Months Initial DOA: Dec. 20, 2019 Initial DOA: Dec.20, 2019 Test Spec. DOA: Sep. 7, 2019 RT ACC DOA: Dec. 20, 2019 RT ACCDescription WLP WLP WLP WLP WLP WLP WLP Appearance CCS CCS CCS CCS CCSCCS CCS pH (65 mg/mL) 9.2-10.2 9.7 9.7 9.7 9.8 9.8 9.8 pH (100 mg/mL)for 130 mg/vial strength % Assay 90.0%-105%   98.2  98.8  97.7  100.0 99.9  99.9  Phenobarbital Sodium Related Substances NMT 0.2% ND ND ND NDND ND 2EPMM PBG NMT 0.2% ND ND ND ND ND ND PEAU NMT 0.2% BQL BQL BQL BQLBQL BQL Any other degradation product NMT 0.2%  0.09 BQL BQL  0.09 BQLBQL Total degradation products NMT 1.5%  0.09 BQL BQL BQL BQL BQL WaterContent by Karl Fischer NMT 4.0% 0.4 NT NP 0.3 NP NP % Residual Solvent(Tertiary To be 1.2 0.6 0.8 4.4 2.9 1.7 butanol) reported Note BookReference 192031-46 192039-9 192031-46 192039-9 to 52 to 23 to 52 to 23LP: White Lyophilized Powder; CCS: Clear Colorless Solution, 1 minuteafter reconstitution; DOA: Date of analysis ND: Not Detected; NP: Notperformed; NMT: Not More Than; BQL: Below Quantitation Limit (0.05%)2EPMM: 2-Ethyl-2-Phenylmalonamide, monohydrate; PBG:α-Phenylbutylguanidine; PEAU: Phenylethylacetylurea

Table 32 depicts results for 65 mg and 130 mg per vial phenobarbitalsodium (from a bulk solution containing 5% isopropanol) stored atstandard and accelerated conditions where under all tested conditions2EPMM, PBG, and PEAU remained undetectable or below quantitation limit(0.05%). Likewise, unspecified degradation products and totaldegradation products remained near or below quantitation limit (0.05).

TABLE 32 (65/130 mg/vial, 5% isopropanol, up to 3 months) Product NamePhenobarbital Sodium for Injection, USP Lyophilized Powder. Bulksolution prepared in 5% Isopropanol Date Manufactured: Feb. 21, 2019Date Initiated: Sep. 5, 2019 Configuration Inverted Stability 25° C.,60% RH (RT) & 40° C., 75% RH (ACC) NPH1966 (65 mg/vial) NPH1968 (130mg/vial) 3 Months 3 Months Initial DOA: Dec. 20, 2019 Initial DOA: Dec.29, 2019 Test Spec. DOA: Sep. 7, 2019 RT ACC DOA: Sep. 7, 2019 RT ACCDescription WLP WLP WLP WLP WLP WLP WLP Appearance CCS CCS CCS CCS CCSCCS CCS pH (65 mg/mL) 9.2-10.2 9.6 9.6 9.7 9.8 9.7 9.7 pH (100 mg/mL)for 130 mg/vial strength % Assay 90.0%-105%   99.7  98.8  100.0  100.0 101.1  99.1  Phenobarbital Sodium Related Substances NMT 0.2% ND ND NDND ND ND 2EPMM PBG NMT 0.2% ND ND ND ND ND ND PEAU NMT 0.2% BQL BQL BQLBQL BQL BQL Any other degradation product NMT 0.2%  0.08 BQL BQL  0.09BQL BQL Total degradation products NMT 1.5%  0.08 BQL BQL BQL BQL BQLWater Content by Karl Fischer NMT 4.0% 0.3 NP NP 0.3 NP NP % ResidualSolvent To be 1.7 0.7 0.7 1.7 1.4 1.4 (Isopropanol) reported Note BookReference 192031-46 192039-9 192031-46 192039-9 to 52 to 23 to 52 to 23WLP: White Lyophilized Powder; CCS: Clear Colorless Solution, 1 minuteafter reconstitution; DOA: Date of analysis ND: Not Detected; NP: Notperformed; NMT: Not More Than; BQL: Below Quantitation Limit (0.05%)2EPMM: 2-Ethyl-2-Phenylmalonamide, monohydrate; PBG:α-Phenylbutyrylguanidine; PEAU: Phenylethylacetylurea

Table 33 depicts initial results for 65 mg and 130 mg per viallyophilized phenobarbital sodium from respective bulk solutionscontaining ethanol or acetonitrile as co-solvent (to obtain 65 mg/vial,0.5 mL of the bulk solution was filled in the vials and for 130 mg/vialstrength, 1 ml of the bulk solution was filled in the vials prior tolyophilization). For all test products, 2EPMM, PBG, and PEAU remainedundetectable or below quantitation limit (0.05%). Notably, unspecifieddegradation products and total degradation products also remainedundetectable or below quantitation limit (0.05%).

TABLE 33 (65/130 mg/vial, acetonitrile or ethanol as co-solvents)Initial Initial NPH22035 NPH22834 Formulation in Formulation inAcetonitrile:Water Ethanol:Water 65 mg 130 mg 65 mg 130 mg Fill volumeFill Volume Fill volume Fill Volume Test Spec. (0.5 mL) (1 mL) (0.5 mL)(1 mL) Description WLP WLP WLP WLP WLP Appearance CCS CCS CCS CCS CCS pH(65 mg/mL) 9.2-10.2 NA NA NA NA % Assay 90.0%-105.0% 103.6  101.2 100.2  98.3 Phenobarbital Sodium Related Substances NMT 0.2% ND ND ND ND2EPMM PBG NMT 0.2% ND ND ND ND PEAU NMT 0.2% BQL BQL BQL BQL Anyunspecified NMT 0.2% ND ND ND ND degradation product Total degradationNMT 1.5% BQL BQL BQL BQL products Water Content by Karl NMT 3.0% 0.4 0.40.2  0.2 Fischer Residual Solvent To be reported NA NA 3.2 19.2 WLP:White Lyophilized Powder; CCS: Clear Colorless Solution, 1 minute afterreconstitution; ND: Not Detected; NMT: Not More Than; BQL: BelowQuantitation Limit (0.05%) 2EPMM: 2-Ethyl-2-Phenylmalonamide,monohydrate; PBG: α-Phenylbutyrylguanidine; PEAU: PhenylethylacetylureaNA: Not available

Notably, the results indicated that the above formulations were alsostorage stable over at least three months as evidenced by all knownimpurities below quantitation limit as shown in Tables 31 and 32.However, an unknown impurity was observed above LOQ level.Interestingly, the residual solvents tertiary butanol and isopropanolwere not completely removed after the first cycle of secondary drying.Tertiary butanol and isopropanol were observed above 0.5% in the 65 mgand 130 mg strength products. The same vials were then subjected toadditional secondary drying at 25° C. for 30 min and 50° C. for 120 min,and the samples were analyzed for residual solvent after the secondcycle of secondary drying.

After the additional secondary drying steps, residual tert-butanol andisopropanol were observed at lower quantities, respectively. As can bereadily seen, the organic solvents could be reduced in quantities, butwere not completely removed, possibly suggesting that the tested organicsolvents could form a bond in the crystal lattice of phenobarbital.However, further process modifications may possibly result in additionalreduction of residual co-solvent, requiring extensive experimentation.Similar formulations were prepared in organic solvents ethanol andacetonitrile. Here as well, the residual solvent ethanol was notcompletely removed, but further process modifications may possiblyresult in additional reduction of residual co-solvent, requiringextensive experimentation.

Organic solvents such as isopropanol, tertiary butanol, ethanol andacetonitrile were investigated as co-solvents during the lyophilization.Two co solvents tertiary butanol and Isopropanol were used asco-solvents at 5%. The 3M stability data for the formulation with IPAand tert-butanol indicates that although no known degradation productswere observed above quantitation limit, the residual solvents could notbe removed completely after lyophilization. When ethanol was used as aco solvent similar results were observed. In view of the fact thatorganic solvents are toxic to all age groups, and in particular toneonates and because residual solvents are typically trapped in thecrystal lattice of the Phenobarbital, the lyophilized product with onlywater as solvent is preferred over formulations containing any organicco solvents.

X-Ray Powder Diffraction. With reference to FIGS. 5A-5D, XRPD wasperformed using the Rigaku Smart-Lab X-ray diffraction system configuredfor reflection Bragg-Brentano geometry using a line source X-ray beam.The X-ray source is a Cu Long Fine Focus tube that was operated at 40 kVand 44 mA. That source provides an incident beam profile at the samplethat changes from a narrow line at high angles to a broad rectangle atlow angles. Beam conditioning slits were used on the line X-ray sourceto ensure that the maximum beam size is less than 10 mm both along theline and normal to the line. The Bragg-Brentano geometry is apara-focusing geometry controlled by passive divergence and receivingslits with the sample itself acting as the focusing component for theoptics. The inherent resolution of Bragg-Brentano geometry is governedin part by the diffractometer radius and the width of the receiving slitused. Typically, the Rigaku Smart-Lab is operated to give peak widths of0.1° 2θ or less. The axial divergence of the X-ray beam is controlled by5.0-degree Soller slits in both the incident and diffracted beam paths.The samples were placed in a low-background, silicon holder using lightmanual pressure to keep the sample surfaces flat and level with thereference surface of the holder. The samples were analyzed from 2 to 40°2θ using a continuous scan of 6° 2θ per minute with an effective stepsize of 0.02° 2θ.

The recitation of ranges of values herein is merely intended to serve asa shorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe full scope of the present disclosure and does not pose a limitationon the scope of the invention otherwise claimed. No language in thespecification should be construed as indicating any non-claimed elementessential to the practice of the claimed invention.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the full scope of the concepts disclosed herein. Thedisclosed subject matter, therefore, is not to be restricted except inthe scope of the appended claims. Moreover, in interpreting both thespecification and the claims, all terms should be interpreted in thebroadest possible manner consistent with the context. In particular, theterms “comprises” and “comprising” should be interpreted as referring toelements, components, or steps in a non-exclusive manner, indicatingthat the referenced elements, components, or steps may be present, orutilized, or combined with other elements, components, or steps that arenot expressly referenced. Where the specification claims refers to atleast one of something selected from the group consisting of A, B, C . .. and N, the text should be interpreted as requiring only one elementfrom the group, not A plus N, or B plus N, etc.

What is claimed is:
 1. A pharmaceutical product, comprising: a containerenclosing a single dose of lyophilized phenobarbital sodium powder;wherein the phenobarbital sodium powder optionally contains residualquantities of tert-butanol, isopropanol, ethanol, or acetonitrile andwherein the phenobarbital sodium powder otherwise does not contain anorganic solvent or preservative; and wherein the phenobarbital sodiumpowder is storage-stable.
 2. The product of claim 1, wherein the singledose phenobarbital sodium powder is a 65 mg dose, a 100 mg dose, a 130mg dose, or a 200 mg dose.
 3. The product of claim 1, wherein thecontainer is a single use glass vial.
 4. The product of claim 1, whereinthe container is a 10 ml vial.
 5. The product of claim 1, wherein thesingle dose, upon reconstitution with water for injection, has a pHrange of between 9.2 and 10.2.
 6. The product of claim 1, wherein thelyophilized phenobarbital sodium powder contains, after storage at 25°C. and 60% relative humidity for 6 months, less than 2.0% water asdetermined by Karl Fischer test.
 7. The product of claim 1, wherein thelyophilized phenobarbital sodium powder contains, after storage at 25°C. and 60% relative humidity for 6 months, less than 1.8% water asdetermined by Karl Fischer test.
 8. The product of claim 1, wherein thelyophilized phenobarbital sodium powder contains, after storage at 25°C. and 60% relative humidity for 6 months, less than 1.6% water asdetermined by Karl Fischer test.
 9. The product of claim 1, wherein thelyophilized phenobarbital sodium powder is storage-stable such that,after storage at 25° C. and 60% relative humidity for 6 months, equal orless than 0.05% of total degradation products from the phenobarbital aredetectable upon reconstitution with water in the reconstituted solution,and wherein the degradation products include 2-ethyl-2-phenylmalonamide(2EPMM), alpha-phenylbutyrylguanidine (PBG), and phenylethylacetylurea(PEAU).
 10. The product of claim 1, wherein the lyophilizedphenobarbital sodium powder is storage-stable such that, after storageat 25° C. and 60% relative humidity for 12 months, equal or less than0.05% of total degradation products from the phenobarbital aredetectable upon reconstitution with water in the reconstituted solution,and wherein the degradation products include 2-ethyl-2-phenylmalonamide(2EPMM), alpha-phenylbutyrylguanidine (PBG), and phenylethylacetylurea(PEAU).
 11. The product of claim 1, wherein the lyophilizedphenobarbital sodium powder is storage-stable such that, after storageat 25° C. and 60% relative humidity for 15 months, equal or less than0.05% of total degradation products from the phenobarbital aredetectable upon reconstitution with water in the reconstituted solution,and wherein the degradation products include 2-ethyl-2-phenylmalonamide(2EPMM), alpha-phenylbutyrylguanidine (PBG), and phenylethylacetylurea(PEAU).
 12. The product of claim 1, wherein the lyophilizedphenobarbital sodium powder is storage-stable such that, after storageat 25° C. and 60% relative humidity for 24 months, equal or less than0.05% of total degradation products from the phenobarbital aredetectable upon reconstitution with water in the reconstituted solution,and wherein the degradation products include 2-ethyl-2-phenylmalonamide(2EPMM), alpha-phenylbutyrylguanidine (PBG), and phenylethylacetylurea(PEAU).
 13. The product of claim 1, wherein the lyophilized amorphousphenobarbital further comprises NaCl.
 14. The product of claim 1,wherein the single dose phenobarbital sodium powder is a 65 mg dose, a100 mg dose, a 130 mg dose, or a 200 mg dose, and wherein the containeris a single use glass vial.
 15. The product of claim 14, wherein thelyophilized phenobarbital sodium powder contains, after storage at 25°C. and 60% relative humidity for 6 months, less than 2.0% water asdetermined by Karl Fischer test.
 16. The product of claim 15, whereinthe lyophilized phenobarbital sodium powder is storage-stable such that,after storage at 25° C. and 60% relative humidity for 6 months, equal orless than 0.05% of total degradation products from the phenobarbital aredetectable upon reconstitution with water in the reconstituted solution,and wherein the degradation products include 2-ethyl-2-phenylmalonamide(2EPMM), alpha-phenylbutyrylguanidine (PBG), and phenylethylacetylurea(PEAU).
 17. The product of claim 15, wherein the lyophilizedphenobarbital sodium powder is storage-stable such that, after storageat 25° C. and 60% relative humidity for 12 months, equal or less than0.05% of total degradation products from the phenobarbital aredetectable upon reconstitution with water in the reconstituted solution,and wherein the degradation products include 2-ethyl-2-phenylmalonamide(2EPMM), alpha-phenylbutyrylguanidine (PBG), and phenylethylacetylurea(PEAU).
 18. The product of claim 1, wherein the lyophilizedphenobarbital sodium powder is lyophilized using a step of freezing to atemperature of about −50° C., a primary drying step at a pressure ofbetween about 50 mTorr and 75 mTorr and at a temperature of betweenabout −50° C. and about 0° C., and a secondary drying step at a pressureof between about 50 mTorr and 75 mTorr and at a temperature of betweenabout 25° C. and about 50° C.
 19. The product of claim 18, wherein thestep of freezing is performed without application of a vacuum.
 20. Theproduct of claim 18, wherein the primary drying step is performed over aperiod of at least 1,000 minutes and/or wherein the secondary dryingstep is performed over a period of at least 200 minutes.